Abstract:
The present invention relates to a method for identifying a DNA breakpoint and agents for use therein. More particularly, the present invention provides a method for identifying a gene translocation breakpoint based on the application of a novel multiplex DNA amplification technique. The method of the present invention facilitates not only the identification of the breakpoint position but, further, enables the isolation of the DNA segment across which the breakpoint occurs. This provides a valuable opportunity to conduct further analysis of the breakpoint region, such as to sequence across this region. The method of the present invention is useful in a range of applications including, but not limited to, providing a routine means to characterize the gene breakpoint associated with disease onset in a patient and thereby enable the design of patient specific probes and primers for ongoing monitoring of the subject disease condition. In addition to monitoring the progression of a condition characterized by the existence of the breakpoint, there is also enabled assessment of the effectiveness of existing therapeutic drugs and/or new therapeutic drugs and, to the extent that the condition is a neoplasm, prediction of the likelihood of a subject&#39;s relapse from a remissive state.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit and priority to International Application No. PCT/AU2008/000779; filed May 30, 2008, which designated the United States and was published in English and claims the benefit of priority to U.S. Provisional No. 60/941,419, filed Jun. 1, 2007. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties. 
    
    
     SEQUENCE LISTING 
     The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled DAVI332 — 001APC.TXT, created May 30, 2008, which is 91 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. 
     FIELD OF THE INVENTION 
     The present invention relates to a method for identifying a DNA breakpoint and agents for use therein. More particularly, the present invention provides a method for identifying a gene translocation breakpoint based on the application of a novel multiplex DNA amplification technique. The method of the present invention facilitates not only the identification of the breakpoint position but, further, enables the isolation of the DNA segment across which the breakpoint occurs. This provides a valuable opportunity to conduct further analysis of the breakpoint region, such as to sequence across this region. The method of the present invention is useful in a range of applications including, but not limited to, providing a routine means to characterise the gene breakpoint associated with disease onset in a patient and thereby enable the design of patient specific probes and primers for ongoing monitoring of the subject disease condition. In addition to monitoring the progression of a condition characterised by the existence of the breakpoint, there is also enabled assessment of the effectiveness of existing therapeutic drugs and/or new therapeutic drugs and, to the extent that the condition is a neoplasm, prediction of the likelihood of a subject&#39;s relapse from a remissive state. 
     BACKGROUND OF THE INVENTION 
     The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 
     Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description. 
     Chromosomal translocations bring the previously unlinked segments of the genome together by virtue of the exchange of parts between non-homologous chromosomes. Although some translocations are not associated with a new phenotype, others may result in disease due to the modulation of protein expression or the synthesis of a new fusion protein. 
     There are two main types of chromosomal translocations which occur, these being reciprocal translocations (also known as non-Robertsonian) and Robertsonian translocations. Further, translocations can be balanced (in an even exchange of material with no genetic information extra or missing) or unbalanced (where the exchange of chromosome material is unequal resulting in extra or missing genes). 
     Reciprocal (non-Robertsonian) translocations usually result in an exchange of material between non-homologous chromosomes and are found in about 1 in 600 newborns. Such translocations are usually harmless and may be found through prenatal diagnosis. However, carriers of balanced reciprocal translocations exhibit an increased risk of creating gametes with unbalanced chromosome translocations thereby leading to miscarriages or children with abnormalities. 
     Robertsonian translocations involve two acrocentric chromosomes that fuse near the centromere region with loss of the short arms. The resulting karyotype has only 45 chromosomes since two chromosomes have fused together. Robertsonian translocations have been observed involving all combinations of acrocentric chromosomes. The most common translocation involves chromosomes 13 and 14 and is seen in about 1 in 1300 persons. Like other translocations, carriers of Robertsonian translocations are phenotypically normal, but exhibit a risk of unbalanced gametes which lead to miscarriages or abnormal offspring. For example, carriers of Robertsonian translocations involving chromosome 21 exhibit a higher probability of having a child with Down syndrome. 
     Diseases which may result from the occurrence of a translocation include:
         (i) Cancer—several forms of cancer are caused by translocations; this mainly having been described in leukemia (eg. acute myelogenous leukemia and chronic myelogenous leukemia).   (ii) Infertility—this can occur where one of the would-be parents carries a balanced translocation, where the parent is asymptomatic but conceived foetuses are not viable.   (iii) Down syndrome—in some cases this is caused by a Robertsonian translocation of about a third of chromosome 21 onto chromosome 14.       

     Specific examples of chromosomal translocations and the disease with which they are associated include:
         t(2;5)(p23;q35)—anaplastic large cell lymphoma   t(8;14)—Burkitt&#39;s lymphoma (c-myc)   t(9;22)(q34;q11)—Philadelphia chromosome, CML, ALL   t(11;14)—Mantle cell lymphoma (Bcl-1)   t(11;22)(q24;q11.2-12)—Ewing&#39;s sarcoma   t(14;18)(q32;q21)—follicular lymphoma (Bcl-2)   t(17;22)—dermatofibrosarcoma protuberans   t(15;17)—acute promyelocytic leukemia (pml and retinoic acid receptor genes)   t(1;12)(q21;p13)—acute myelogenous leukemia   t(9;12)(p24;p13)—CML, ALL (TEL-JAK2)   t(X;18)(p11.2;q11.2)—Synovial sarcoma   t(1;11)(q42.1;q14.3)—Schizophrenia   t(1;19)—acute pre-B cell leukemia (PBX-1 and E2A genes).       

     The shorthand t(A;B)(p1;q2) is used to denote a translocation between chromosome A and chromosome B. The information in the second set of parentheses, when given, gives a precise location within the chromosome for chromosomes A and B respectively—with p indicating the short arm of the chromosome, q indicating the long arm, and the numbers of p and q refers to regions, bands and sub-bands seen when staining the chromosomes under microscope. 
     As detailed above, chronic myelogenous leukemia is an example of a neoplastic condition which is caused by a chromosomal translocation. However, unlike many neoplastic conditions, its treatment prospects are quite good if it can be effectively diagnosed and monitored. 
     In virtually all cases of chronic myelogenous leukemia, a specific translocation is seen. This translocation involves the reciprocal fusion of small pieces from the long arms of chromosome 9 and 22. The altered chromosome 22 is known as the Philadelphia chromosome (abbreviated as Ph1). When the breakpoint of the Ph1 chromosome was sequenced, it was found that the translocation creates a fusion gene by bringing together sequences from the c-ABL proto-oncogene and another BCR (breakpoint cluster region). The BCR-ABL fusion gene encodes a phosphoprotein (p210) that functions as a dysregulated protein tyrosine kinase and predisposes the cell to become neoplastic. This hypothesis is supported by finding that expression of p210 results in transformation of a variety of hematopoietic cell lines in vitro and that mice transgenic for the human BCR-ABL gene develop a number of hematologic malignancies. 
     Another well studied example of a translocation generating cancer is seen in Burkitt&#39;s lymphoma. In some cases of this B cell tumor, a translocation is seen involving chromosome 8 and one of three other chromosomes (2, 14 or 22). In these cases, a fusion protein is not produced. Rather, the c-myc proto-oncogene on chromosome 8 is brought under transcriptional control of an immunoglobulin gene promoter. In B cells, immunoglobulin promoters are transcriptionally quite active, resulting in over expression of c-myc, which is known from several other systems to exhibit monogenic properties. Accordingly, this translocation results in aberrant high expression of an oncogenic protein. 
     The classical method of diagnosing chromosomal translocations, such as those observed in chronic myelogenic leukemia, is by karyotyping. For many translocations, however, it is now possible to detect the translocation by PCR, using primers which span the breakpoint. In some cases, the PCR technique can also be used for sensitive detection and monitoring of treatment. Monitoring to determine the effect of treatment has become increasingly important for diseases such as chronic myeloid leukemia and acute promyelocytic leukemia as increasingly effective treatment has been developed. For monitoring in these 2 diseases, the starting material for the PCR is RNA. The translocation breakpoint is within the introns of the respective genes and, as a consequence, RNA splicing removes the sequence of RNA transcribed by introns and results in only one or a very limited number of final mRNA products being produced, despite the very large number of different translocations which are present in the patient population. 
     However, the use of RNA as the starting material to detect and quantify the translocation by PCR suffers the disadvantage that RNA is a difficult molecule to work with due to its inherent susceptibility to degradation. DNA is a more stable molecule. However, the initial identification and characterisation of the breakpoint in the context of DNA is much more difficult since cluster regions of chromosomal fusion sites often span large introns of several tens of thousands of nucleotides. These sizes are too large for direct coverage by a single PCR reaction. There therefore exists an ongoing need to develop means for routinely conducting breakpoint analyses on DNA samples. 
     In work leading up to the present invention, a novel multiplex amplification reaction has been developed which enables the localisation and analysis of a breakpoint in a DNA sample. Despite the precise position of the breakpoint being unknown, the method of the present invention nevertheless enables diagnosis of the existence of the breakpoint in a DNA sample and the isolation and analysis of the breakpoint region using a relatively modest and simple multiplex amplification reaction. The design of this amplification reaction results in the advantage that generation of long PCR products is not required. Still further, the optional incorporation of a primer hybridisation tag region at the 5′ end of the amplification primers enables the rapid generation of large copy numbers of the amplicons generated using these primers and therefore facilitates the isolation and analysis of the amplicons. 
     SUMMARY OF THE INVENTION 
     Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 
     As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 
     The subject specification contains nucleotide sequence information prepared using the programme PatentIn Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator &lt;210&gt; followed by the sequence identifier (eg. &lt;210&gt;1, &lt;210&gt;2, etc). The length, type of sequence (DNA, etc) and source organism for each sequence is indicated by information provided in the numeric indicator fields &lt;211&gt;, &lt;212&gt; and &lt;213&gt;, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1, SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field &lt;400&gt; in the sequence listing, which is followed by the sequence identifier (eg. &lt;400&gt;1, &lt;400&gt;2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as &lt;400&gt;1 in the sequence listing 
     One aspect the present invention is directed to a method of identifying a gene breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the other reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i); 
           
         
       
    
     The present invention therefore preferably provides a method of identifying a chromosomal gene translocation breakpoint, said method comprising: 
     (i) contacting a genomic DNA sample with: 
     
         
         
           
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
           
         
       
    
     There is therefore preferably provided a method of identifying a gene breakpoint, said method comprising: 
     (i) contacting a DNA sample with 
     
         
         
           
             (a) one to thirty forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one to thirty forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
           
         
       
    
     The present invention therefore provides a method of identifying a gene translocation breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one to thirty forward primers directed to a DNA region of the antisense strand of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (i)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (i)(b);
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one to thirty forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (iii)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (iii)(b); 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
           
         
       
    
     According to this preferred embodiment there is provided a method of identifying a chromosomal BCR-ABL translocation breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one or more forward primers directed to a DNA region of BCR or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of ABL or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one or more forward primers directed to a DNA region of BCR or fragment thereof, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to ABL or fragment thereof, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
           
         
       
    
     The present invention therefore preferably provides a method of identifying a chromosomal BCR-ABL translocation breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one to thirty forward primers directed to a DNA region of BCR or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of ABL or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (i)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (i)(b);
 
(ii) amplifying the DNA sample of step (i);
 
(iii) contacting the amplicon generated in step (ii) with:
 
             (a) one to thirty forward primers directed to a DNA region of BCR or fragment thereof, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of ABL or fragment thereof, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (iii)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (iii)(b); 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) isolating and sequencing said amplified DNA.
 
           
         
       
    
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic representation of the strategy for amplification of the breakpoint region in the first round PCR. The forward primers for gene A and the reverse primers for gene B are preferably used in pools rather than individually. Only primer pairs which closely straddle the breakpoint will produce efficient amplification. The tags and tag primers are not shown. The strategy for the second round PCR is the same although the forward and reverse primers are just internal to their corresponding primers in the first round. In the case of chronic myeloid leukemia, gene A is the BCR gene and gene B is the ABL gene. Primer binding sites are staggered so that the maximum amplicon size does not exceed 1 kilobase. 
         FIG. 2  is a schematic representation of a protocol for isolation of the BCR-ABL translocation breakpoint in chronic myeloid leukemia. 
         FIG. 3  is an image of the results of electrophoresis showing amplified material from study of one patient. NFA was the pool of 6 forward BCR primers and NFA 13 and NFA 14 were 2 pools each containing 12 reverse ABL primers. NFA 13/14 was a pool containing the 24 ABL primers belonging to pools 13 and 14. 
         FIG. 4  is a representation of the sequences of the breakpoints in 4 patients with chronic myeloid leukemia. The numbers on the left are the Genbank base numbers for the BCR and ABL genes. 
         FIG. 5  shows the site of the DNA breakpoints in the ABL and BCR genes in the 27 patients with breakpoints isolated and identified. Blue regions in the ABL gene represent the exons 1a, 1b and E2. Red regions in the BCR gene represent exons 13, 14 and 15. 
       Isolation of the BCR-ABL Breakpoint in Chronic Myeloid Leukemia (CML) 
       Samples from 29 CML patients have been studied using the invention. In 27 of these patients the breakpoint sequences have been isolated and detailed sequencing information obtained. For one patient it has not been possible to amplify the BCR/ABL breakpoint. For the remaining patient a suspected breakpoint has been amplified. Sequence information shows the BCR gene at the 5′ end and ABL sequence at the 3′ end, however this breakpoint has not been confirmed with primers made specifically for the suspected regions. 
         FIG. 6  is a comparison of DNA-based and RNA-based quantification of minimal residual disease (MRD) in samples of blood from 16 patients with CML. ND=not detected. Y-axis shows the number of leukemic cells as a proportion of total cells. The DNA-based PCR used patient-specific primers synthesised using knowledge of the breakpoint sequence in the patient being studied, the RNA-based PCR was the conventional approach using reverse transcription followed by PCR using generic primers. Black symbols show MRD detected by both techniques, red symbols show disease detected only by DNA-PCR and blue symbols show disease not detected. DNA-based PCR appears to be approximately 2 orders of magnitude more sensitive than RNA-based PCR. 
         FIG. 7  is an illustration of the isolation of the PML-RARα breakpoint from a sample from the one patient with acute promyelocytic leukemia 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is predicated, in part, on the determination that gene translocation breakpoints can be routinely and easily identified, via DNA analysis, by sequentially performing two PCR reactions which use multiple primers directed to the genes flanking the breakpoint which are themselves tagged at their 5′ end with a DNA region suitable for use as a primer hybridisation site. The simultaneous use of multiple primers facilitates the performance of a short PCR, rather than the long PCRs which have been performed to date. By sequentially performing a second PCR using primers directed to gene regions internal to those used in the first reaction, amplification of a DNA molecule spanning the breakpoint region can be achieved in a manner which enables the identification and isolation of a smaller amplification product than has been enabled to date in terms of the analysis of genomic DNA. By incorporating unique tag regions which can themselves be targeted by a primer, amplification of the initial amplicon can be rapidly achieved, thereby overcoming any disadvantage associated with the use of a low concentration of starting primer directed to the genes flanking the breakpoint. The method of the present invention therefore provides a simple yet accurate means of identifying and analysing a gene breakpoint using DNA. To this end, it would be appreciated that although the method of the present invention is exemplified by reference to chronic myelogenic leukemia, this method can be applied to any situation in which a gene breakpoint is sought to be identified via a DNA sample. 
     Accordingly, in one aspect the present invention is directed to a method of identifying a gene breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the other reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
           
         
       
    
     It should be understood that in a preferred embodiment of the present invention, where one primer is used in step (i)(a), it is preferable that two or more primers are used in step (i)(b). The converse applies where one primer is used in step (i)(b). Similarly, in another preferred embodiment, where one primer is used in step (iii)(a), it is preferable that two or more primers are used in step (iii)(b). The converse applies where one primer is used in step (iii)(b). 
     Reference to the “flanking genes” 5′ and 3′ to the breakpoint should be understood as a reference to the genes or gene fragments on either side of the breakpoint. In terms of the 5′ and 3′ nomenclature which is utilised in the context of these genes/gene fragments, this should be understood as a reference to the 5′? 3′ orientation of the sense strand of double stranded DNA from which the DNA of interest derives. Accordingly, reference to “the flanking gene 5′ to the breakpoint” should be understood as a reference to the sense strand of double stranded DNA. To this end, any reference to “gene” or “gene fragment” herein, to the extent that it is not specified, is a reference to the sense strand of double stranded DNA. Reference to the forward primer being directed to the antisense strand of the flanking gene 5′ to the breakpoint therefore indicates that the forward primer bears the same DNA sequence as a region of the sense strand 5′ to the breakpoint and therefore will bind to and amplify the antisense strand corresponding to that region. 
     Reference to “gene” should be understood as a reference to a DNA molecule which codes for a protein product, whether that be a full protein or a protein fragment. In terms of chromosomal DNA, the gene will include both intron and exon regions. However, to the extent that the DNA of interest is cDNA, such as might occur if the DNA of interest is vector DNA, there may not exist intron regions. Such DNA may nevertheless include 5′ or 3′ untranslated regions. Accordingly, reference to “gene” herein should be understood to encompass any form of DNA which codes for a protein or protein fragment including, for example, genomic DNA and cDNA. 
     Reference to a gene “breakpoint” should be understood as a reference to the point at which a fragment of one gene recombines with another gene or fragment thereof. That is, there has occurred a recombination of two genes such that either one or both genes have become linked at a point within one or both of the genes rather than the beginning or end of one gene being linked to the beginning or end of the other gene. That is, at least one of the subject genes has been cleaved and has recombined with all or part of another gene. The recombination of the two non-homologous gene regions may occur by any method including but not limited to chromosomal gene translocations or in vitro homologous recombinations (such as may occur where a DNA segment is being inserted into a vector or an artificial chromosome or where a vector portion thereof chromosomally integrates in a host cell). 
     Preferably, the subject gene breakpoint is a chromosomal gene translocation breakpoint. As detailed hereinbefore, chromosomal gene translocations are known to occur and, in some cases, lead to the onset of disease states. Since a gene translocation between two genes will not necessarily result in the breakpoint occurring at precisely the same nucleotide position on the two genes each time the translocation event occurs, it is not possible to assume that the breakpoint position in one patient, such as the Philadelphia chromosome breakpoint in one CML patient, will be the same in another patient. The method of the present invention enables the simple yet accurate determination of a gene breakpoint using DNA. 
     The present invention therefore preferably provides a method of identifying a chromosomal gene translocation breakpoint, said method comprising: 
     (i) contacting a genomic DNA sample with: 
     
         
         
           
             (a) one or more forward primers directed to a DNA region of the flanking gene fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one or more forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
           
         
       
    
     Reference to “DNA” should be understood as a reference to deoxyribonucleic acid or derivative or analogue thereof. In this regard, it should be understood to encompass all forms of DNA, including cDNA and genomic DNA. The nucleic acid molecules of the present invention may be of any origin including naturally occurring (such as would be derived from a biological sample), recombinantly produced or synthetically produced. 
     Reference to “derivatives” should be understood to include reference to fragments, homologs or orthologs of said DNA from natural, synthetic or recombinant sources. “Functional derivatives” should be understood as derivatives which exhibit any one or more of the functional activities of DNA. The derivatives of said DNA sequences include fragments having particular regions of the DNA molecule fused to other proteinaceous or non-proteinaceous molecules. “Analogs” contemplated herein include, but are not limited to, modifications to the nucleotide or nucleic acid molecule such as modifications to its chemical makeup or overall conformation. This includes, for example, modification to the manner in which nucleotides or nucleic acid molecules interact with other nucleotides or nucleic acid molecules such as at the level of backbone formation or complementary base pair hybridisation. The biotinylation or other form of labelling of a nucleotide or nucleic acid molecules is an example of a “functional derivative” as herein defined. 
     As detailed hereinbefore, the method of the present invention is predicated on the use of multiple oligonucleotide primers to facilitate the multiplexed amplification of a DNA sample of interest. In one embodiment of the present invention, the DNA sample of interest is a hybrid gene which comprises a portion of one gene (gene A) which is located 5′ to the translocation breakpoint and a second gene (gene B) which is located 3′ to the translocation breakpoint. In a particular embodiment, gene A is BCR and gene B is ABL. The identification of the existence and nature of a gene translocation breakpoint is achieved by using two or more forward primers directed to gene A and two or more reverse primers directed towards gene B. The primers directed to gene A are designed to hybridise at intervals along gene A and the primers directed to gene B are similarly designed to hybridise at intervals along gene B. In the first round PCR, the primers which will amplify the hybrid gene are the upstream primers which hybridise to that portion of gene A which lies 5′ to the breakpoint and the downstream primers which hybridise to that portion of gene B which lies 3′ to the breakpoint. Furthermore, since small amplicons are amplified more efficiently than larger amplicons, there will occur selection for amplification directed by the primer pair which hybridises closest to the breakpoint. The same principle holds for the second round primers and, since in one embodiment each second round primer corresponds to an individual first-round primer but hybridises internal to it with regard to the breakpoint, there will be further selection for amplification by the pair of the second round primers which bound the breakpoint. Without limiting the present invention in any way, the second round of PCR amplification provides additional specificity for amplification of the breakpoint region. Following the second round PCR, successful amplification of the sequence surrounding the breakpoint will be evident as a band of amplified material on electrophoresis. 
     Since it is not known precisely where the breakpoint lies, it is possible that one or more of the internal primers may not hybridise to their target region sequence due to this sequence having been effectively spliced out during the translocation event. However, in one embodiment, the forward and reverse primers selected for the first round amplification are directed to amplifying from the 5′ and 3′ end regions, respectively, of the gene fragments flanking the breakpoint. The second round primers are then directed to internal regions of the gene fragments flanking the breakpoint, that is, the regions which are closer to the breakpoint than the regions targeted by the first round primers. Again, it would be appreciated that since the precise location of the breakpoint is not known, one or more of these forward and/or reverse primers may not hybridise to the DNA sample due to their target region sequence having been spliced out. In terms of the second round “internal primers”, it should be understood that this is a reference to a population of primers of which at least one primer, but preferably all the primers, are designed to amplify the subject DNA from a point which, when considered in the context of the translocated gene itself (rather than the antisense strand or the amplification product), is 3′ of the most 3′ of the forward primers used in the first round amplification and 5′ of the most 5′ of the reverse primers used in the first round amplification. By using the approach of a two step amplification using progressively more internally localised primers, amplification of DNA spanning the breakpoint region can be achieved without the requirement to perform long PCRs or to generate very long and cumbersome amplification products. 
     Reference to a “primer” or an “oligonucleotide primer” should be understood as a reference to any molecule comprising a sequence of nucleotides, or functional derivatives or analogues thereof, the function of which includes hybridisation to a region of a nucleic acid molecule of interest (the DNA of interest also being referred to as a “target DNA”) and the amplification of the DNA sequence 5′ to that region. It should be understood that the primer may comprise non-nucleic acid components. For example, the primer may also comprise a non-nucleic acid tag such as a fluorescent or enzymatic tag or some other non-nucleic acid component which facilitates the use of the molecule as a probe or which otherwise facilitates its detection or immobilisation. The primer may also comprise additional nucleic acid components, such as the oligonucleotide tag which is discussed in more detail hereinafter. In another example, the primer may be a protein nucleic acid which comprises a peptide backbone exhibiting nucleic acid side chains. preferably, said oligonucleotide primer is a DNA primer. 
     Reference to “forward primer” should be understood as a reference to a primer which amplifies the target DNA in the DNA sample of interest by hybridising to the antisense strand of the target DNA. 
     Reference to “reverse primer” should be understood as a reference to a primer which amplifies the target DNA in the DNA sample of interest and in the PCR by hybridising to the sense strand of the target DNA. 
     The design and synthesis of primers suitable for use in the present invention would be well known to those of skill in the art. In one embodiment, the subject primer is 4 to 60 nucleotides in length, in another embodiment 10 to 50 in length, in yet another embodiment 15 to 45 in length, in still another embodiment 20 to 40 in length, in yet another embodiment 25 to 35 in length. In yet still another embodiment, primer is about 26, 27, 28, 29, 30, 31, 32, 33 or 34 nucleotides in length. Without limiting the invention in any way, the primers are designed in one embodiment to have a T M  of 65 to 70° C. This enables the PCR to use a high annealing temperature, which minimises non-specific annealing and amplification. Each forward or reverse primer for the second round PCR is designed to hybridise to a sequence which is close, either downstream for the forward primer or upstream for the reverse primer, to the hybridisation sequence for its corresponding forward or reverse first-round primer. Designing the corresponding primers to hybridise to closely adjoining sequences minimises the probability that the translocation breakpoint will involve or occur between the hybridisation sequences. even if this does occur, the sequence surrounding the translation breakpoint can still be amplified by the immediately upstream or downstream, as the case may be, primer pair. 
     In the exemplified embodiment described herein, primers were chosen so that their binding sites were staggered with the separation between adjacent binding sites being approximately 500 bases. This was done so that the amplified material would have range in size, up to a maximum length of approximately 1 kilobase. This strategy is in contrast to the strategy of “Long PCR” which would require fewer primers and a less complex multiplex PCR reaction. The advantages of the strategy of the present invention are that the standard shorter PCR reaction is more robust and the amplified product can be sequenced immediately rather than requiring another set of PCR reactions to break it up into smaller amplicons which are suitable for sequencing. 
     In terms of the number of primers which are used in the method of the invention, this can be determined by the person of skill in the art. With regard to the total number of primers, the variables which require consideration are the size of the gene region which is being targeted and the distance between the sequences to which the primers hybridise. In order to amplify PCR fragments which are no larger than about 1 kb, the primers can be designed to hybridise at intervals of approximately 500 bases. With regard to CML, nearly all BCR translocations involve one of two regions, each of approximately 3 kb in length. In this case, 12 outer forward primers and 12 corresponding inner primers may be used. The ABL gene, however, is larger, approximately 140 kb in length, and up to 280 outer reverse primers and 280 inner reverse primers may be used. In one particular embodiment, a combination of 6 forward primers and 24 reverse primers is used and in another embodiment a combination of 6 forward primers and 140 reverse primers. The primer number which is selected to be used will depend on the genes involved in the translocation and thus may vary from translocation to translocation and will involve consideration of the competing issues of the number of PCR reactions which are required to be performed versus the probability of generating non-specific products during a PCR reaction. As would be understood by the person of skill in the art, a large number of primers in each individual PCR reaction decreases the number of PCR reactions but increases the probability of non-specific amplification reactions. 
     In one embodiment, the method of the present invention is performed using at least three primers, in another embodiment at least four primers. In yet another embodiment said invention is performed using 6-10 primers, 6-15 primers, 6-20 primers, 6-25 primers or 6-30 primers. In still another embodiment there is used 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 primers. 
     There is therefore preferably provided a method of identifying a gene breakpoint, said method comprising: 
     (i) contacting a DNA sample with 
     
         
         
           
             (a) one to thirty forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one to thirty forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
preferably, said gene breakpoint is a gene translocation breakpoint and still more preferably a chromosomal gene translocation breakpoint.
 
           
         
       
    
     The primers which are used in the method of the present invention are of a relatively low individual concentration due to the starting primer pool comprising multiple individual primers. This reduces the risk of inducing inhibition of PCR. In order to facilitate a successful amplification result, it is therefore necessary to enable the generation of sufficient amplicons for detection and isolation. In one aspect of the present invention, this can be facilitated by tagging the primers with an oligonucleotide which can be used as a primer hybridisation site. In addition to the primers directed towards genes A and B, each PCR reaction may therefore also contain concentrations of two oligonucleotides which are directed to the tag, as a primer hybridisation site. These oligonucleotide sequences act as primers and enable efficient secondary amplification of the amplicons generated by the initial hybridisation and extension of the primers directed towards genes A and B. In one embodiment, the primer which is directed to the tag exhibits a T M  of 65° C.-70° C. in order to minimise non-specific amplification. Thus these primers are directed towards overcoming the potential problem posed by the low concentrations of the primers directed towards A and B. Nevertheless, in some situations it may not be necessary to use one or both tag primers. For example, when there are only six forward primers for the BCR gene each primer may be at a concentration which is sufficient for relatively efficient amplification. Still further, it should be appreciated that the oligonucleotide tags provide an additional use when they are present in the final PCR round, since the tag primers can also be used for sequencing. Accordingly, although the tag is suitable for use as a site for primer hybridisation, it should be understood that the subject tag may also be useful for other purposes, such as a probe binding site in the context of Southern gel analysis or to enable isolation of the primer or the amplicon extended therefrom. To this end, the tag may comprise a non-nucleic acid component, such as a protein molecule or biotin which would enable isolation, for example by affinity chromatography, streptavidin binding or visualisation. 
     In order to ensure that these tags do not interfere with the extension of the primer, the primers are linked to the oligonucleotide tag at their 5′ end. Reference to “oligonucleotide tag” should therefore be understood as a reference to a nucleotide sequence of less than 50 nucleotides which is linked to the 5′ end of the forward and reverse primers of the present invention. In one embodiment, the tag is 25-30 bases in length. It should also be understood that consistently with the definitions provided in relation to the forward and reverse primers, the oligonucleotide tags herein described may also comprise non-nucleic acid components such as isolation or visualisation tags eg. biotin, enzymatic labels, fluorescent labels and the like. This enables quick and simple isolation or visualisation of the tagged primers or amplicons via non-molecular methods. 
     That the oligonucleotide tag is “operably linked” to the primer should be understood as a reference to those regions being linked such that the functional objectives of the tagged primer, as detailed hereinbefore, can be achieved. In terms of the means by which these regions are linked and, further, the means by which the subject oligonucleotide primer binds to its target DNA region, these correspond to various types of interactions. In this regard, reference to “interaction” should be understood as a reference to any form of interaction such as hybridisation between complementary nucleotide base pairs or some other form of interaction such as the formation of bonds between any nucleic or non-nucleic acid portion of the primer molecule or tag molecule with any other nucleic acid or non-nucleic acid molecule, such as the target molecule, a visualisation means, an isolation means or the like. This type of interaction may occur via the formation of bonds such as, but not limited to, covalent bonds, hydrogen bonds, van der Wals forces or any other mechanism of interaction. preferably, to the extent that the interaction occurs between the primer and a region of the target DNA, said interaction is hybridisation between complementary nucleotide base pairs. In order to facilitate this interaction, it is preferable that the target DNA is rendered partially or fully single stranded for a time and under conditions sufficient for hybridisation with the primer to occur. 
     Without limiting the present invention to any one theory or mode of action, the inclusion of an oligonucleotide tag which can itself function as a primer hybridisation site can assist in facilitating the convenient and specific amplification of the amplicon generated by the forward and reverse primers of the present invention. Accordingly, this overcomes somewhat the amplification limitation which is inherent where a relatively low starting concentration of the forward and reverse primers is used. Where the starting concentration of forward and reverse primers is sufficiently high, it may not be necessary to use a tag. Accordingly, in a preferred embodiment, the DNA sample of interest is contacted with both the forward and reverse primers of the present invention and primers directed to the oligonucleotide tags of the forward and reverse primers such that the amplification reaction of step (ii) proceeds in the context of all these primers. It should be understood, however, that although it is preferred that amplification based on both the gene primers and the tag primers is performed simultaneously, the method can be adapted to perform the tag primer based amplification step after the completion of the gene primer based amplification. 
     The DNA sequence of the tags may be the same or different. With respect to a first round amplification, the tags may be the same if the purpose is to amplify the initial amplification product. However, if one wishes to selectively enrich for amplicons containing the sequence of one of the flanking genes, the primer directed to the tag region of the primer of the gene of interest (eg. gene A) should differ to the primer directed to the tag region of the primer of the other gene (eg. gene B). In another example, in terms of a second or subsequent round of amplification, the tags which are used for sequencing would be required to be different to prevent the simultaneous sequencing of both strands. 
     The present invention therefore provides a method of identifying a gene translocation breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one to thirty forward primers directed to a DNA region of the antisense strand of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ relative to the gene breakpoint, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (i)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (i)(b);
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one to thirty forward primers directed to a DNA region of the flanking gene or fragment thereof located 5′ relative to the gene breakpoint, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of the flanking gene or fragment thereof located 3′ to the gene breakpoint, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (iii)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (iii)(b); 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
preferably said gene translocation breakpoint is a chromosomal gene translocation breakpoint.
 
           
         
       
    
     It should be understood that the oligonucleotide primers and tags of the present invention should not be limited to the specific structure exemplified herein (being a linear, single-stranded molecule) but may extend to any suitable structural configuration which achieves the functional objectives detailed herein. For example, it may be desirable that all or part of the oligonucleotide is double stranded, comprises a looped region (such as a hairpin bend) or takes the form of an open circle confirmation, that is, where the nucleotide primer is substantially circular in shape but its terminal regions do not connect. 
     Facilitating the interaction of the nucleic acid primer with the target DNA may be performed by any suitable method. Those methods will be known to those skilled in the art. 
     Methods for achieving primer directed amplification are also very well known to those of skill in the art. In a preferred method, said amplification is polymerase chain reaction, NASBA or strand displacement amplification. Most preferably, said amplification is polymerase chain reaction. To this end, in one embodiment of the invention, a 20 minute hybridisation provides good amplification in the first round PCR. 
     Reference to a “sample” should be understood as a reference to either a biological or a non-biological sample. Examples of non-biological samples includes, for example, the nucleic acid products of synthetically produced nucleic acid populations. Reference to a “biological sample” should be understood as a reference to any sample of biological material derived from an animal, plant or microorganism (including cultures of microorganisms) such as, but not limited to, cellular material, blood, mucus, faeces, urine, tissue biopsy specimens, fluid which has been introduced into the body of an animal and subsequently removed (such as, for example, the saline solution extracted from the lung following lung lavage or the solution retrieved from an enema wash), plant material or plant propagation material such as seeds or flowers or a microorganism colony. The biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy sample may require homogenisation prior to testing or it may require sectioning for in situ testing. Further, to the extent that the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilise the sample. 
     To the extent that the target DNA is present in a biological sample, the biological sample may be directly tested or else all or some of the nucleic acid material present in the biological sample may be isolated prior to testing. It is within the scope of the present invention for the target nucleic acid molecule to be pre-treated prior to testing, for example inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing). 
     Reference to “contacting” the sample with the primer should be understood as a reference to facilitating the mixing of the primer with the sample such that interaction (for example, hybridisation) can occur. Means of achieving this objective would be well known to those of skill in the art. 
     The choice of what type of sample is most suitable for testing in accordance with the method disclosed herein will be dependent on the nature of the situation, such as the nature of the condition being monitored. For example, in a preferred embodiment a neoplastic condition is the subject of analysis. If the neoplastic condition is a lymphoid leukemia, a blood sample, lymph fluid sample or bone marrow aspirate would likely provide a suitable testing sample. Where the neoplastic condition is a lymphoma, a lymph node biopsy or a blood or marrow sample would likely provide a suitable source of tissue for testing. Consideration would also be required as to whether one is monitoring the original source of the neoplastic cells or whether the presence of metastases or other forms of spreading of the neoplasia from the point of origin is to be monitored. In this regard, it may be desirable to harvest and test a number of different samples from any one mammal. Choosing an appropriate sample for any given detection scenario would fall within the skills of the person of ordinary skill in the art. 
     The term “mammal” to the extent that it is used herein includes humans, primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, deer, foxes). preferably, the mammal is a human or a laboratory test animal. Even more preferably the mammal is a human. 
     As detailed hereinbefore, in one embodiment the method of the present invention is performed as a sequential two step amplification using multiple second round primers each of which is directed to a gene region which is either 3′ (for the forward primers) or 5′ (for the reverse primers) to that which is targeted by the corresponding first round primers. The person of skill in the art would appreciate that in some cases it may not be necessary to conduct a second round amplification. The necessity to perform a second round amplification may also be obviated if a selective or enrichment step as described below is performed. This situation may arise when the sequence around the breakpoint is amplified very efficiently and there is very little non-specific amplification such that a clearly defined band of amplification product is observed on electrophoresis of the product of the first round amplification or if the subsequent selection step is very efficient. In general, however, it is expected that a sequential two step amplification process would be used in order to minimise non-specific amplification and to generate a relatively short amplification product which spans the breakpoint region. In general, it is expected that the amplification product would be less than 1.5 kb, less than 1 kb, less than 0.8 kb or less than 0.5 kb. It should be understood that depending on the size of the genes which have been translocated, the method of the invention may be adapted to incorporate third or fourth round amplification steps in order to further minimise non-specific amplification. This can be an issue owing to the number of primers present in the multiplexed reaction and to the fact that one of the genes participating in the translocation often contains multiple repetitive sequences such as Alu. Nevertheless, it is expected that the need for further rounds of amplification would be unlikely. 
     Although the method of the present invention has been designed such that the amplification steps can be sequentially performed directly on the amplification product of a previous amplification, this should not be understood as a limitation in terms of whether any additional steps are sought to be incorporated by the skilled person, such as enrichment/selection steps. For example, one may seek to select for the desired amplicons after the first round amplification and to thereafter conduct the second round amplification on their material alone. Methods which one could utilise to select or enrich include:
     (i) a selection step based on the unique oligonucleotide tags which are linked to the primers. Accordingly, since the tags themselves are also amplified and therefore form part of the amplicon, they could be used as a probe site to enable isolation of amplicons which are the result of both forward and reverse primer amplification and therefore should span the breakpoint. Alternatively, biotinylation of one of the tags provides means of identifying and isolating amplicons which have resulted from extension by either the forward or reverse primers. For example, by flooding the amplification product with biotinylated primer, the primer can act as a probe to identify the amplicons of interest and the biotinylation can provide a basis for isolating those amplicons. By ensuring that each of the primer groups of the present invention comprises a unique tag, it is possible to select out, with significant particularity, only specific amplicons of interest. In particular, the skilled person would seek to exclude amplicons which have been amplified by a forward primer but which have not then been amplified by a reverse primer, thereby indicating that the subject amplicon possibly does not extend across the breakpoint. By selecting out the amplicons which are most likely spanning the breakpoint, a subsequent round of amplification is more specifically targeted and less likely to generate unwanted amplicons as a result of either inherent cross-hybridisation of primers or the amplification of amplicons which do not flank both sides of the breakpoint.   (ii) One may seek to run the products on a gel and excise out only certain bands or regions which are likely to be relevant and thereafter subject these to a further amplification step. When a band is present on the gel after the second round amplification, if there are any problems in sequencing an attempt can be made to clean it up by cutting the product out of the gel and performing a series of PCR reactions using individual primers and/or smaller pools of primers. For example, one might use individual forward BCR primers and pools containing only 12 reverse ABL primers.   (iii) one may expose the amplified products to one or more rounds of bottleneck PCR in order to provide negative selection against non-specific amplified products.   

     Without limiting the application of the present invention to any one theory or mode of action, in a classical PCR, the primers and reaction conditions are designed so that primer hybridisation and extension of the forward and reverse primers occur at or close to the maximum efficiency so that the number of amplicons approximately doubles with each cycle resulting in efficient exponential amplification. Bottleneck PCR, however, is predicated on the use of forward and reverse primer sets where the primers of one set have been designed or are otherwise used under conditions wherein they do not hybridise and extend efficiently. Accordingly, although the efficient primer set will amplify normally, the inefficient set will not. As a consequence, when a sequence of interest is amplified, the number of amplicon strands is significantly less than that which would occur in a classical PCR. Efficient amplification only commences once amplicons have been generated which incorporate, at one end, the tag region of the inefficient primer. At this point, the primers directed to the tag regions effect a normal amplification rate. A “bottleneck” is therefore effectively created in terms of the generation of transcripts from the inefficient primer set. 
     A more severe bottleneck is usefully created where the inefficient primers are directed to commonly repeated sequences, such as an alu sequence. Amplification of unwanted product may result if such binding sites are closely apposed and if the inefficient primers can act as forward primers and reverse primers. However, owing to both primers being inefficient, amplification is initially extremely inefficient and there is a severe bottleneck. Efficient amplification only commences once amplicon strands have been generated which comprise the tag region of the inefficient primer at one end and its complement at the other. After any given number of cycles, the number of such amplicons is, however, substantially less than that which occurs during amplification of the sequence of interest. The amount of unwanted product at the end of the amplification reaction is correspondingly reduced. 
     Hybridisation and extension of an inefficient primer which has correctly hybridised to the sequence of interest followed in a subsequent cycle by hybridisation and extension of an efficient primer to the previously synthesised amplicon generates a template to which the tag primer can efficiently hybridise and extend. Since such molecules together with their complements provide upstream and downstream binding sites, each for an efficient primer (the tag primer and one member of the efficient set), succeeding cycles of amplification from such templates are both efficient and exponential. The result is that, after an initial lag or “bottleneck”, the overall rate of amplification speeds up in later cycles so that a near doubling of amplicon number with each cycle results. However, the net result is that there is negative selection against amplification of undesired amplicons as compared to amplicons of the sequence of interest, owing to the bottleneck at each end for the former and only at one end for the latter. 
     Accordingly, if the same number of commencing target sequences is considered and comparison to the amplification produced by classical PCR is made, application of the bottleneck PCR will produce a lesser increase in the number of amplicons of the sequence of interest and an even lesser increase in the number of amplicons of unwanted sequences. Although amplification of both wanted and unwanted products occurs, there is relative enrichment of the sequence of interest relative to the unwanted sequences. There is an inverse relationship between absolute amplification and enrichment since decreasing the efficiency of the inefficient primer set produces increased enrichment at the expense of lesser amplification. 
     Once the amplification rounds have been completed, the amplicons spanning the breakpoint region can be analysed. In a preferred embodiment, the subject amplicon is isolated by excision of a gel band containing that amplicon and sequenced in order to characterise the breakpoint region. To the extent that a band excised from a gel is to be analysed, it may be necessary to further amplify the DNA contained therein in order to provide sufficient material for sequencing. The oligonucleotide tags hereinbefore described provide a suitable primer hybridisation site to facilitate further amplification of the isolated amplicons. 
     As detailed hereinbefore, the method of the present invention provides a simple and routine means of identifying and characterising any breakpoint region, such as the nature, accuracy and stability of a site directed insertion of a gene into a chromosome or vector (this being important in the context of gene therapy), but in particular the chromosomal gene translocation breakpoints that are characteristic of many diseases. Examples of such translocations and diseases include, but are not limited to:
         t(2;5)(p23;q35)—anaplastic large cell lymphoma   t(8;14)—Burkitt&#39;s lymphoma (c-myc)   t(9;22)(q34;q11)—Philadelphia chromosome, CML, ALL (BCR-ABL recombination)   t(11;14)—Mantle cell lymphoma (Bcl-1)   t(11;22)(q24;q11.2-12)—Ewing&#39;s sarcoma   t(14;18)(q32;q21)—follicular lymphoma (Bcl-2)   t(17;22)—dermatofibrosarcoma protuberans   t(15;17)—acute promyelocytic leukemia (pml and retinoic acid receptor genes)   t(1;12)(q21;p13)—acute myelogenous leukemia   t(9;12)(p24;p13)—CML, ALL (TEL-JAK2)   t(X;18)(p11.2;q11.2)—Synovial sarcoma   t(1;11)(q42.1;q14.3)—Schizophrenia   t(1;19)—acute pre-B cell leukemia (PBX-1 and E2A genes).
 
preferably, said chromosomal gene translocation is a BCR-ABL translocation or a PML-RARalpha translocation.
       

     According to this preferred embodiment there is provided a method of identifying a chromosomal BCR-ABL translocation breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one or more forward primers directed to a DNA region of BCR or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to a DNA region of ABL or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags;
 
(ii) amplifying the DNA sample of step (i);
 
(iii) optionally contacting the amplicon generated in step (ii) with:
 
             (a) one or more forward primers directed to a DNA region of BCR or fragment thereof, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; and 
             (b) one or more reverse primers directed to ABL or fragment thereof, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) analysing said amplified DNA.
 
preferably, said amplification steps are performed using 1-30 forward primers and 24-300 reverse primers.
 
           
         
       
    
     In terms of the embodiment of the invention exemplified herein, primers were chosen so that their binding sites were staggered with the separation between adjacent binding sites being approximately 500 bases. This was done so that the amplified material would have range in size, up to a maximum length of approximately 1 kilobase. This strategy may be contrasted to the prior art strategy of “Long PCR” which would require fewer primers and a less complex multiplex PCR reaction. One of the advantages of the strategy of the present invention is that the standard shorter PCR reaction is more robust and the amplified product can be sequenced immediately rather than requiring another set of PCR reactions to break it up into smaller amplicons which are suitable for sequencing. 
     The present invention therefore preferably provides a method of identifying a chromosomal BCR-ABL translocation breakpoint, said method comprising: 
     (i) contacting a DNA sample with: 
     
         
         
           
             (a) one to thirty forward primers directed to a DNA region of BCR or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of ABL or fragment thereof, which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (i)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (i)(b);
 
(ii) amplifying the DNA sample of step (i);
 
(iii) contacting the amplicon generated in step (ii) with:
 
             (a) one to thirty forward primers directed to a DNA region of BCR or fragment thereof, which primers are directed to DNA regions which are located 3′ to one or more of the forward primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (b) twenty-four to four hundred reverse primers directed to a DNA region of ABL or fragment thereof, which primers are directed to DNA regions which are located 5′ to one or more of the reverse primers of step (i) and which primers are optionally operably linked at their 5′ end to an oligonucleotide tag; 
             (c) a primer directed to the forward primer oligonucleotide tag of step (iii)(a); and 
             (d) a primer directed to the reverse primer oligonucleotide tag of step (iii)(b); 
             wherein the oligonucleotide tags of the forward primers are the same relative to the forward primer tags of step (iii)(a) and the oligonucleotide tags of the reverse primers are the same relative to the reverse primer tags of step (iii)(a) but which forward primer oligonucleotide tags are different relative to the reverse primer tags and which forward and reverse primer tags of step (iii) are different relative to the forward and reverse primer tags of step (i);
 
(iv) amplifying the DNA sample of step (iii);
 
(v) isolating and sequencing said amplified DNA.
 
           
         
       
    
     More preferably, said DNA sequence is a blood derived sample. 
     The method of the present invention has broad application including, but not limited to:
     (i) enabling the design and generation of patient specific probes which can be used for the ongoing monitoring of a patient who is diagnosed with a disease condition characterised by chromosomal gene translocation. Results obtained by this means for chronic myeloid leukemia are shown in  FIG. 6 .   (ii) the analysis and monitoring of in vitro and in vivo gene transfection systems which are directed to integrating a gene or other DNA region into a chromosome, vector, plasmid, artificial chromosome or the like. Where the general site at which recombination should occur is known, the present invention can be applied to determine the specific point and nature of the integration (i.e. the breakpoint). It can also be used to monitor the ongoing stability of the genetic recombination event by virtue of enabling the generation of specific primers.   

     Accordingly, in yet another aspect there is provided a method of monitoring a disease condition in a mammal, which disease condition is characterised by a gene breakpoint, said method comprising screening for the presence of said breakpoint in a biological sample derived from said mammal, which breakpoint has been identified in accordance with the method hereinbefore defined. 
     Methods of screening for the subject breakpoint would be well known to those skilled in the art and include any suitable probe-based screening technique, such as PCR based methods. By virtue of the identification of the breakpoint region in accordance with the method of the invention, one can design an appropriate probe set to specifically amplify the subject breakpoint. 
     In one embodiment, said gene breakpoint is a chromosomal gene translocation breakpoint such as:
         t(2;5)(p23;q35)   t(8;14)   t(9;22)(q34;q11)   t(11;14)   t(11;22)(q24;q11.2-12)   t(14;18)(q32;q21)   t(17;22)   t(15;17)   t(1;12)(q21;p3)   t(9;12)(p24;p13)   t(X;18)(p11.2;q11.2)   t(1;11)(q42.1;q14.3)   t(1;19).       

     In another embodiment, said condition is:
         anaplastic large cell lymphoma   Burkitt&#39;s lymphoma   CML, ALL   Mantle cell lymphoma   Ewing&#39;s sarcoma   follicular lymphoma   dermatofibrosarcoma protuberans   acute promyelocytic leukemia   acute myelogenous leukemia   Synovial sarcoma   Schizophrenia; or   acute pre-B cell leukemia.       

     Still another aspect of the present invention is directed to a DNA primer set, which primer set is designed to amplify and/or otherwise detect a gene breakpoint, which breakpoint has been identified in accordance with the method hereinbefore defined. 
     The present invention is now described by reference to the following non-limiting examples and figures. 
     Example 1 
     Isolation of BCR/ABL Breakpoint Product from gDNA of Patient 1 
     
         
         
           
             Genomic DNA extracted by Qiagen Flexigene kit 
             1 st  Round PCR (50 ng genomic DNA)—all reactions performed in duplicate 
           
         
       
    
     Forward primer pool—FA (Contains 7 forward BCR primers BCRF1-BCRF7 each with same 5′ tag sequence (A), Total 50 ng (7.14 ng each) 
     Reverse primer pool—R3/4 (Pool of 24 oligonucleotide reverse ABL primers, each with same 5′ tag sequence (C), Total 50 ng (2.08 ng each) 
     Forward and reverse tag sequence primers (A,C)—25 ng of each 
     PCR Conditions 
     1×PCR buffer, 5 mM MgCl 2 , 0.75 ul dUTP (300 uM each), 0.4 ul Platinum Taq (2 U) 
     Cycling Conditions 
     95/4 min 
     (97° C./1 min, 65° C./20 min, 72° C./1 min)×5 
     (96° C./30 sec, 65° C./20 min, 72° C./1 min)×5 
     (92° C./30 sec, 65° C./20 min, 72° C./1 min)×10
         2 nd  Round PCR (1 st  round reaction diluted 1/200 in sterile water)       

     Forward primer pool—NFA (Contains 7 forward internal BCR primers BFN1-BFN7 each with same 5′ tag sequence (B), Total 50 ng (7.14 ng each) 
     Reverse primer pool—RN3/4 (Pool of 24 oligonucleotide reverse internal ABL primers, each with same 5′ tag sequence (D), Total 50 ng (2.08 ng each) 
     Forward and reverse tags (B,D)-25 ng of each 
     PCR Conditions 
     1×PCR buffer, 5 mM MgCl 2 , 0.75 ul dUTP (300 uM), 0.4 ul Pt Taq (2 U) 
     Cycling Conditions 
     95/4 min 
     (94° C./30 sec, 65° C./10 min, 72° C./1 min)×10 
     (94° C./30 sec, 65° C./5 min, 72° C./1 min)×15
         PCR products (7 ul) resolved on 1.5% (v/v) agarose gel at 120 volts
 
Identification of BCR/ABL Breakpoint from Patient 1
   PCR products resolved on 1.5% (v/v) agarose gel at 120 volt       

     Band excised and purified via Flexigene kit
         Reamplification of bands by PCR (1/1000 dilution of purified product)       

     Forward primer—Tag B (25 ng) 
     Reverse primer—TagD (25 ng) 
     PCR Conditions 
     1×PCR buffer, 5 mM MgCl 2 , 0.75 ul dUTP (300 uM), 0.4 ul Pt Taq (2 U) 
     Cycling Conditions 
     95/4 min 
     (94° C./30 sec, 65° C./30 sec, 72° C./30 sec)×35
         PCR Product Sequenced with TagB Primer (Flinders Sequencing Facility)
 
Confirmation of Breakpoint by PCR
   PCR performed on gDNA (50 ng) across breakpoint       

     Patient 1 gDNA vs 10× Normal gDNA (several primer combinations) 
     Forward primer—BCR (patient specific) (25 ng) 
     Reverse primer—ABL (patient specific) (25 ng) 
     PCR Conditions 
     1×PCR buffer, 5 mM MgCl 2 , 0.75 ul dUTP (300 uM), 0.4 ul Pt Taq (2 U) 
     Cycling Conditions 
     95/4 min 
     (97° C./1 min, 65° C./30 sec, 72° C./30 sec)×5 
     (96° C./30 sec, 65° C./30 sec, 72° C./30 sec)×5 
     (92° C./30 sec, 65° C./30 sec, 72° C./30 sec)×25
         PCR products resolved on 3% (v/v) agarose gel at 120 volt       

     Band excised and purified via Qiagen minElute kit 
     Products sequenced with 5′ BCR specific primer to confirm BCR/ABL breakpoint (Flinders sequencing facility). 
     Nearly all translocations involve a 3 kb region of the BCR gene and 140 kb region of the ABL gene. Six forward primers used to cover the region of the BCR gene and 282 primers used to cover the region of the ABL gene. Six PCRs are set up, each containing one of the BCR primers, all of the ABL primers, and the common tag primer. 
     If necessary, a second round of PCR is performed with a nested internal BCR primer and 282 nested internal ABL primers Alternatively, 1-3 rounds of Bottleneck PCR are performed in order to remove non-specific amplified products and reveal the amplified translocation sequence. 
     The ABL gene is very rich in Alu sequences, and the BCR gene also contains one such sequence. The ABL primers have therefore undergone a selection procedure which sequentially involves, for each ABL primer:
         design using standard criteria   pairing with each BCR primer and testing by electronic PCR for amplification off the BCR template. Primers that fail this criterion are discarded.   incorporation in a pool of 12 or 24 ABL primers, pairing the pool with each BCR primer, and testing by experimental PCR using a BCR template which has been previously produced by PCR amplification. Any pool that that produces amplification and thus fails this test is further analysed by testing each of the individual ABL primers to determine which is responsible for amplification. When identified, this primer is discarded.       

     The BCR and ABL primers used in Example 1 are shown in Example 2. 
     Example 2 
     Primers Used for Isolation of BCR-ABL Translocation Breakpoint in Chronic Myeloid Leukemia 
     BCR Primers 
     
       
         
               
               
               
             
           
               
                   
                 1st Rd 
                   
               
               
                   
                 BCRF1-FT0 
                 cttctccctgacatccgtgg 
               
               
                   
               
               
                   
                 BCRF2-FT0 (-5) 
                 acacagcatacgctatgcacatgtg 
               
               
                   
               
               
                   
                 BCRF3-FT0 
                 gaggttgttcagatgaccacgg 
               
               
                   
               
               
                   
                 BCRF4-FT1 (-10) 
                 cagctactggagctgtcagaacag 
               
               
                   
               
               
                   
                 BCRF5-FT0 
                 tgggcctccctgcatcc 
               
               
                   
               
               
                   
                 BCRF6-FT0 
                 tccccctgcaccccacg 
               
               
                   
               
               
                   
                 2nd Rd 
                   
               
               
                   
                 BCRF1-FT1 
                 tgacatccgtggagctgcagatgc 
               
               
                   
               
               
                   
                 BCRF2-FT1 
                 acatgtgtccacacacaccccacc 
               
               
                   
               
               
                   
                 BCRF3-FT1 
                 accacgggacacctttgaccctgg 
               
               
                   
               
               
                   
                 BCRF4-FT1 (-4) 
                 ctggagctgtcagaacagtgaagg 
               
               
                   
               
               
                   
                 BCRF5-FT1 
                 tccctgcatccctgcatctcctcc 
               
               
                   
               
               
                   
                 BCRF6-FT1 
                 cccacgacttctccagcactgagc 
               
             
          
         
       
     
     The second round primers were internal to the first round primers and were used either for a second round together with internal ABL primers or for performing Bottleneck PCR in order to eliminate non-specific amplified material and facilitate isolation of the translocation breakpoint. 
     Various combinations of the forward and reverse primers can be used. In one embodiment, the protocol that was used was to set up 6 PCRs, each containing a different BCR primer and all 282 ABL primers 
     282 Reverse ABL Primers Used for the First PCR Round and the Tag Sequence which was on the 5′ End of Each Primer 
                                 Tag A   gcaacactgtgacgtactggagg                   R1   gtctatctaaaattcacaaggaatgc                   R2   aggcaaagtaaaatccaagcaccc                   R3   cactcctgcactccagcctgg                   R4   caaccaccaaagtgcttttcctgg                   R5   atatggcatctgtaaatattaccacc                   R6   tgcctcggcctcccaaagtgc                   R7   agccaccacacccagccagg                   R8   aataactgttttctccccccaaaac                   R9   tgttttacaaaaatggggccatacc                   R10   acttaagcaaattctttcataaaaaggg                   R11   ctttcaattgttgtaccaactctcc                   R12   acctcctgcatctctccttttgc                   R13   aaataaagttttgagaaccataagtgg                   R14   caccatcacagctcactgcagc                   R15   aacctctttgagaatcggatagcc                   R16   aaataaagtacatacctccaattttgc                   R17   gacacattcctatgggtttaattcc                   R18   tgtaaaatatggtttcagaagggagg                   R19   gcaggtggataacgaggtcagg                   R20   ccagccaagaatttcaaagattagc                   R21   gaagggagatgacaaagggaacg                   R22   gcagaagaactgcttgaacctgg                   R23   gtggtcccagctactcgagagg                   R24   ccctcagcaaaactaactgaaaagg                   R25   tagaaaccaagatatctagaattccc                   R26   ccacgcccggcggaataaatgc                   R27   acaaaaaaagaggcaaaaactgagag                   R28   ctgggcgcagtggctcatgcc                   R29   tggctgtgaggctgagaactgc                   R30   ctgggcgacagagtgagactcc                   R31   aagtctggctgggcgcagtgg                   R32   aatggacaaaagaggtgaactggc                   R33   gatagagtgaaaacgcacaatggc                   R34   aattaaacagctaggtcaatatgagg                   R35   ggtctccactatcaagggacaag                   R36   aagcagctgttagtcatttccagg                   R37   aggcatcctcagattatggctcc                   R38   cctgagtaacactgagaccctgc                   R39   aacactcaagctgtcaagagacac                   R40   attcaggccaggcgcagtggc                   R41   taaatcgtaaaactgccacaaagc                   R42   cagaggagtaggagaaggaaaagg                   R43   ggtagctatctaccaagtagaatcc                   R44   atcagattggaaaaagtcccaaagc                   R45   ctcctgaaaagcacctactcagc                   R46   ctccttaaacctgaggtactggg                   R47   ttttctcctaatagaccaccattcc                   R48   ctgctgtattaccatcactcatgtc                   R49   ctggccaacatagtgaaaccacg                   R50   atttgaataggggttaaagtatcattg                   R51   cacttcagtggaagttggcatgc                   R52   gtttttcttcgaagtgataaacatacg                   R53   gctccttagtctatgtacctgtgg                   R54   tactctggcatggtaactggtgc                   R55   acaaaggactaggtctgtggagc                   R56   ccaagtttaccaaattaccaaagttacc                   R57   tgagccgatatcacgccactgc                   R58   tcccaataaaggttttggcccagg                   R59   ctgggtagcaaattagggaacagg                   R60   ctggccagaaaagacagttttatcc                   R61   ggttcccaggaagggataacacc                   R62   tcactccaggaggttccatttcc                   R63   aggcttggaaataagcagcagtgg                   R64   attcatacaatggaatactactcagc                   R65   taagtgatcctcccacctcaacc                   R66   tataagaggaagactggggctgg                   R67   tcatacttatgcaggttataggagg                   R68   caagatcacgccactgcactcc                   R69   aaaataaatagctggtgctcaagatc                   R70   caccagcctcattcaacagatgg                   R71   caatgcagcctcaacctcctgg                   R72   gttaggtcaggtgctcatgtctg                   R73   aagtttcaaaaggacatgtacaaaatg                   R74   tcctgaagaggctgcagcttcc                   R75   ctggtgcacattcccaagtgtgc                   R76   catgttggccatgttcttctgagg                   R77   ctcagcctcccgagtagctgg                   R78   aaagacatttaagaggagatgaggc                   R79   tgctgggattacaggcgtgagc                   R80   tgtgacttccatccgcagctcc                   R81   gacacttttgtggagctttcatgg                   R82   catgtgagggggcacgtcttgc                   R83   tcttctctatgagaaaagtggttgc                   R84   tggcaaaatgctatcgagctgcc                   R85   tatgaacacagccggcctcagg                   R86   gaggttgcagtgagctgagatcg                   R87   gtcaagcacccagtccgatacc                   R88   atctgggcttggtggcgcacg                   R89   gttaagcgggtcccacatcagc                   R90   cagccagtttcagtagaaagatgc                   R91   gacccaagcataaggggactagc                   R92   cccaaaaagtttacaagagaaattttc                   R93   cgcctgtagtcccagctactcg                   R94   cgcgtgatgcggaaaagaaatcc                   R95   tctactatgaaccctccttcagac                   R96   gtgctgggattacaggtgtgagc                   R97   ttatccaaatgtcccagggcagg                   R98   ctgccagcactgctcgccagc                   R99   gctactgcaggcagtgccttcc                   R100   catccaagcccaaggtgtcagg                   R101   tgtttgcatgtaatttcaggaagcc                   R102   gatccgtcactgttaacactcagg                   R103   ctcacagtcacaagctcctgagc                   R104   gagatgatgctggggtcacagg                   R105   ttagaagaatgggatcgcaaagg                   R106   cggtattcaaatatgaggtcaggc                   R107   gtaaatcctgctgccagtcttcc                   R108   acagggtcagacagagccttgg                   R109   agttattgatctaactatacaacaagc                   R110   aaagactaggggccggggacg                   R111   ctggtagaaataaagacaacaaagcc                   R112   gtgccaagtaattaaaagtttgaaacc                   R113   ggcttttgaagggagcaccacc                   R114   gaaggataaatacctatgatactttcc                   R115   ggcagggaaatactgtgcttcaag                   R116   gtggtgaaattccacctcagtacc                   R117   tcccaaagtgctgggattacagg                   R118   gaaattagcaaacaatgccaagacg                   R119   taagtattggaccgggaaggagg                   R120   ctatcattttgctcaaagtgtagcc                   R121   atttcacaaactacagaggccagg                   R122   tagacttctgtctctctatgctgc                   R123   tgagtgagctgccatgtgatacc                   R124   acttcacaccagcctgtccacc                   R125   taactcatatcctcagagagaccc                   R126   agaggttcctcgattcccctgc                   R127   gtgtcagcgtcccaacacaaagc                   R128   gaaagtggatgggcaagcattgc                   R129   gtgatcacctcacagctgcagg                   R130   gtttgtttagtcaaggcatttcacc                   R131   cctcagcctccagagtagctgg                   R132   taaaagaaaactcctccttcctgg                   R133   aatgtgctatgtctttaaatccatgg                   R134   agctggcaaatctggtaatataaaag                   R135   gcttgaacctggaaggtggagg                   R136   gcaggcatgctaagaccttcagc                   R137   cagctccatgaataactccacagg                   R138   gcttgaacccaggaggcagagg                   R139   atcgaagatgccactgcaagagg                   R140   ccaaccacacttcaggggatacc                   R141   cacgccagtccactgatactcac                   R142   gggtttcaccatgttggccagg                   R143   cccaacaaaggctctggcctgg                   R144   atgacagcagaggagcttcatcc                   R145   gcaggctacgagtaaaaggatgg                   R146   cgggtaaaatcttgcctccttcc                   R147   aaacttaaaccaatggtggatgtgg                   R148   agagactgaggaactgttccagc                   R149   gaaacggtcttggatcactgatcc                   R150   tgcgcatgatatcttgtttcaggg                   R151   ggcctccgtttaaactgttgtgc                   R152   gaatgctggcccgacacagtgg                   R153   tcttggtatagaaaagccagctgg                   R154   gcaaaagcccaagagcccctgg                   R155   ttctcccaaaatgagccccaagg                   R156   gtggtgacgtaaacaaaaggtacc                   R157   gcaaattccatgtgaatcttattggc                   R158   cctgatctatggaacagtggtgg                   R159   gttacaaacgttgcagtttgcaacg                   R160   gaaccccgtcaacagtgatcacc                   R161   acaggacctcaaggcaaggagc                   R162   catacctaaaatagaaatgtctatccc                   R163   gagttgcatatatgttttataaatccc                   R164   tgagcccacatccataaagttagc                   R165   accgcaacctttgccgcctgg                   R166   taaatattttgtatggagtcaccacc                   R167   aaagccaggagaaaaagttatgagg                   R168   tcccaaagtcccaggattacagg                   R169   tcactatggagcatctccgatgg                   R170   agttccctggaagtctccgagg                   R171   aaaataatcacccagcccacatcc                   R172   acaaaactacagacacagaaagtgg                   R173   tttgggaggctgaggtaggtgg                   R174   aaagacagtgaaacatctataaggg                   R175   cattttgggagaccagggcagg                   R176   gcatgggacagacacaaagcagc                   R177   gaataacaaagagagccggctgg                   R178   taaaccttttattgaaaattgtcaaatgg                   R179   cgcctcagcctcccaaagtgc                   R180   tacattagttttataggtccagtagg                   R181   gaaggtttattcatattaaaatgtgcc                   R182   ctggcttctgtggtttgagttgg                   R183   acagacctacctcctaaggatgg                   R184   gctagcttttgtgtgtaagaatggg                   R185   ggcctactcacacaatagaatacc                   R186   gcaccattgcactccagcctgg                   R187   gaaattaggataaaggttgtcacagc                   R188   cagaagtgttcaaggtgaaactgtc                   R189   ctgaatcatgaaatgttctactctgc                   R190   tgtcaacttgactgggccatacg                   R191   ctcccgtatagttgggattatagg                   R192   gcttggagttccttgaaattcttgg                   R193   cctggtggctccagttttctacc                   R194   aactcctgacctcatgatccacc                   R195   gctgggattacaggcatgagcc                   R196   ttctcctttatccttggtgacattc                   R197   tcccaaagtgctgggattacagg                   R198   gtcataagtcagggaccatctgc                   R199   ctgtttcattgatttccagactggc                   R200   gcaatctcggctcactgcaagc                   R201   gaagaagtgactatatcagatctgg                   R202   ttcaccatgttggccaggctgg                   R203   catcactgaagatgacaactgagc                   R204   gtccagcctgggcgatagagc                   R205   gaggaaagtctttgaagaggaacc                   R206   ggtacactcaccagcagttttgc                   R207   gagcaactggtgtgaatacatatgg                   R208   caatacctggcaccacatacacc                   R209   gggactacaggcatgtgccacc                   R210   cggtggctcacgcgtgtaatcc                   R211   caactgttaaatctctcatggaaacc                   R212   gacaaaggattagaaatgcaccc                   R213   ggaaatgttctaaaactggattgtgg                   R214   aataataatagccaggtgtggtagc                   R215   ctggaacactcacacattgctgg                   R216   ctgggtgacagagcgagactcc                   R217   cccaaatcatccccgtgaaacatgc                   R218   gaccctgcaatcccaacactgg                   R219   ctctcaggccttcaaactacacc                   R220   caggaaagggctcgctcagtgg                   R221   atctgcaaaagcagcagagcagg                   R222   gtacccatgacagacaagttttagg                   R223   cttatcccctactgtctcctttgg                   R224   ggatggtctcgatctcctgacc                   R225   aggttagagaccttcctctaatgc                   R226   agctgggattacaggtgcctgc                   R227   gctgaggcaggttggggctgc                   R228   acatttaacgtctcctaacttctcc                   R229   gtgctgcgattacaggtgtgagc                   R230   tatgacagcagtattatactatcacc                   R231   ctggggaccaaatctgaactgcc                   R232   gtagctattgttatttccaaaagagg                   R233   gcttgggaccccaggacaagg                   R234   cctggccaacatggggaaatcc                   R235   aattgcttgaacctgggaggtgg                   R236   gcctaagacccaaaagctattagc                   R237   catattaaagggccatattcaaattgg                   R238   ggatgtaaccagtgtatatcacagg                   R239   ggaagtttagtccacatcttctagc                   R240   gcacccacaggacaaccacacg                   R241   gggacgcgcctgttaacaaagg                   R242   gggctgggggccacgctcc                   R243   cgcaaaagtgaagccctcctgg                   R244   gaaatcctacttgatctaaagtgagc                   R245   tttgagcaacttggaaaaaataagcg                   R246   ttcccaaaagacaaatagcacttcc                   R247   ccattttgaaaatcacagtgaattcc                   R248   gaaaagaaaaccctgaattcaaaagg                   R249   tgctgaaaagaagcatttaaaagtgg                   R250   ctcttaccagtttcagagctttcc                   R251   ttttcagccaaaaatcaaggacagg                   R252   cttgagcccaggagtttgagacc                   R253   cgcctgtagtaccctctactagg                   R254   ggtaaagaaagaaggatttgaaaacc                   R255   taagagtaatgaggttaaagtttatgc                   R256   catttttattgtcacaggccatttgc                   R257   gccacgccttctcttctgccacc                   R258   tgcctctcctgactgcactgtg                   R259   ccatgctctaccacgcccttgg                   R260   cattcaggctggagtgcggtgg                   R261   cttaaaaattgtctggctaagacattg                   R262   ttgctcttgttgcccgggttgg                   R263   gagcttagaggaaaagtattatttcc                   R264   tggtgctgtgccagacgctgg                   R265   cagatctttttggctattgtcttgg                   R266   gaaggaaagggcctcccactgc                   R267   catgaaaaagcatgctggggagg                   R268   caaacataaaaaagctttaatagaagcc                   R269   tcccaactatgaaaaaatagaagacg                   R270   cacaaattagccgggcatggtgg                   R271   cttcctttactgagtctttctaaagc                   R272   tgtcctttgaaatgtaggtatgtgg                   R273   ggatcttgcaatactgacatctcc                   R274   atttgaaaagaactgaaggatctacc                   R275   gtgagctgagatctcgtctctgc                   R276   tttgtctgaaacagattctaaaagttgg                   R277   gcaggtgcctgtagtcccagc                   R278   gtttgagcttctaaaattcatggattc                   R279   gtggtaggtcaaaccgcaattcc                   R280   accaaatcagacatatcagctttgg                   R281   cacagaacggatcctcaataaagg                   R282   gttaactcctcccttctctttatgg            
282 Reverse ABL Primers Used for the Second PCR Round and the Tag Sequence which was on the 5′ End of Each Primer
 
     
       
         
               
               
               
             
           
               
                   
                 2nd Round 
                   
               
               
                   
                 Tag D 
                 gtgttcagagagcttgatttccagg 
               
               
                   
               
               
                   
                 RN1 
                 cccacttgatttttcccacatgg 
               
               
                   
               
               
                   
                 RN2 
                 atttatttagatgaagtgaatattttcc 
               
               
                   
               
               
                   
                 RN3 
                 atttagtttgtttaactgtgagtgc 
               
               
                   
               
               
                   
                 RN4 
                 gtacagaagtgcttgatgcatacc 
               
               
                   
               
               
                   
                 RN5 
                 aggcagataaaaattctccattagc 
               
               
                   
               
               
                   
                 RN6 
                 acaagcacgagccacagcacc 
               
               
                   
               
               
                   
                 RN7 
                 cgctcttgttgcccaggctgg 
               
               
                   
               
               
                   
                 RN8 
                 cccaaaacagactttctagataacc 
               
               
                   
               
               
                   
                 RN9 
                 ttcaaattgctttttttctactcacc 
               
               
                   
               
               
                   
                 RN10 
                 gatctgaaaaaagtgacaggttgg 
               
               
                   
               
               
                   
                 RN11 
                 cactgaaatttgaaaggaacatatgg 
               
               
                   
               
               
                   
                 RN12 
                 tctggtgcagtggcctctagg 
               
               
                   
               
               
                   
                 RN13 
                 accataagtggttttacctgatgg 
               
               
                   
               
               
                   
                 RN14 
                 cccaggcgcaggtgattctcc 
               
               
                   
               
               
                   
                 RN15 
                 ggtggctcacgcctgaaatcc 
               
               
                   
               
               
                   
                 RN16 
                 cacagtccacgtgccacaatcc 
               
               
                   
               
               
                   
                 RN17 
                 aatcatgttaacacatccctctcc 
               
               
                   
               
               
                   
                 RN18 
                 gaagagagtgttgaaaggttaagc 
               
               
                   
               
               
                   
                 RN19 
                 cgagaccatactggctaagatgg 
               
               
                   
               
               
                   
                 RN20 
                 attagccacacaataaatgttctgg 
               
               
                   
               
               
                   
                 RN21 
                 tttgaaaagcgttgcaatatgatgc 
               
               
                   
               
               
                   
                 RN22 
                 ggttgcagtgagccgagatcg 
               
               
                   
               
               
                   
                 RN23 
                 ggtgggaggactgcctgagc 
               
               
                   
               
               
                   
                 RN24 
                 aacagagagaaaaaacacaaattacc 
               
               
                   
               
               
                   
                 RN25 
                 gatatctagaattcccaaatacttgg 
               
               
                   
               
               
                   
                 RN26 
                 gtgatagaattaaaggaaaaaataaacg 
               
               
                   
               
               
                   
                 RN27 
                 attgttccttttctaaatattctacc 
               
               
                   
               
               
                   
                 RN28 
                 cagcactttgggaggctgagg 
               
               
                   
               
               
                   
                 RN29 
                 cacagaggtttcacagtgctgg 
               
               
                   
               
               
                   
                 RN30 
                 aacttctgcttctgtccataatgc 
               
               
                   
               
               
                   
                 RN31 
                 gcctgtaatcccagcactttgg 
               
               
                   
               
               
                   
                 RN32 
                 gccagtaaacatatgaaaaggtgc 
               
               
                   
               
               
                   
                 RN33 
                 aattatgtaaataaagagtgaaaagg 
               
               
                   
               
               
                   
                 RN34 
                 cccctacacagaaaaaacaattcc 
               
               
                   
               
               
                   
                 RN35 
                 tgagtgtcaaagaaaaatacaattgg 
               
               
                   
               
               
                   
                 RN36 
                 atacacagagaaaatgagtccacc 
               
               
                   
               
               
                   
                 RN37 
                 aacactccccttctctgtttagc 
               
               
                   
               
               
                   
                 RN38 
                 gatattctttgcaacctaggatgc 
               
               
                   
               
               
                   
                 RN39 
                 ctctaaaactaatcagcaatgtaacc 
               
               
                   
               
               
                   
                 RN40 
                 cacctgtaatcccagcactttgg 
               
               
                   
               
               
                   
                 RN41 
                 cgtaaaactgccacaaagcttgtagg 
               
               
                   
               
               
                   
                 RN42 
                 gtggcagaggtgcaagcaagc 
               
               
                   
               
               
                   
                 RN43 
                 acagaaatgacaaacgcatgtacc 
               
               
                   
               
               
                   
                 RN44 
                 acactctcttagctaggctttgg 
               
               
                   
               
               
                   
                 RN45 
                 gagcttggaatagggcagttcc 
               
               
                   
               
               
                   
                 RN46 
                 ctgggttctttaaacatgtccagg 
               
               
                   
               
               
                   
                 RN47 
                 tcaagaaaggacactgcagtggc 
               
               
                   
               
               
                   
                 RN48 
                 catgcacacaaactatctcattcc 
               
               
                   
               
               
                   
                 RN49 
                 tagccgggcatggtggcacg 
               
               
                   
               
               
                   
                 RN50 
                 atcatgctgattgaatttcaaatagc 
               
               
                   
               
               
                   
                 RN51 
                 ttggcatgcagggcagtgacc 
               
               
                   
               
               
                   
                 RN52 
                 ggtggtgagataataacacctgc 
               
               
                   
               
               
                   
                 RN53 
                 ttgctatataataatcatttgtgatcc 
               
               
                   
               
               
                   
                 RN54 
                 cggtaactgttactctgggatgg 
               
               
                   
               
               
                   
                 RN55 
                 aggctaggttcccttctcttcc 
               
               
                   
               
               
                   
                 RN56 
                 gtagtgcctagcacagagaaagc 
               
               
                   
               
               
                   
                 RN57 
                 ctagcctgggcaacaagagcg 
               
               
                   
               
               
                   
                 RN58 
                 tctctctcctctctgggatcag 
               
               
                   
               
               
                   
                 RN59 
                 gtttgaatatttgtatgcagcaagc 
               
               
                   
               
               
                   
                 RN60 
                 tagaacaaattctggcttataaaagc 
               
               
                   
               
               
                   
                 RN61 
                 ccactctacctttattccttgcc 
               
               
                   
               
               
                   
                 RN62 
                 agaccagaatatgcaagcagagg 
               
               
                   
               
               
                   
                 RN63 
                 ggacgttttgctggtgtctgcg 
               
               
                   
               
               
                   
                 RN64 
                 aaggaacaaactgttgtcacatgc 
               
               
                   
               
               
                   
                 RN65 
                 atgtagctgggactacaggtgc 
               
               
                   
               
               
                   
                 RN66 
                 ggctcatgcctgtaatcccagc 
               
               
                   
               
               
                   
                 RN67 
                 atgaggttttcacacaaaaagatgc 
               
               
                   
               
               
                   
                 RN68 
                 tgggcgacagagcaagactcc 
               
               
                   
               
               
                   
                 RN69 
                 aaatgtccctaaaagtgatcaacagc 
               
               
                   
               
               
                   
                 RN70 
                 cagactcagttttacctcatcagc 
               
               
                   
               
               
                   
                 RN71 
                 agtgatctttcctctttaacctcc 
               
               
                   
               
               
                   
                 RN72 
                 ccagctattcaggaggccaagg 
               
               
                   
               
               
                   
                 RN73 
                 cttaaacattatgacactgtcttgc 
               
               
                   
               
               
                   
                 RN74 
                 ccaggtctatgaggccgttcc 
               
               
                   
               
               
                   
                 RN75 
                 tccaaagcatccctacattatacc 
               
               
                   
               
               
                   
                 RN76 
                 acatacatacatgcagtgactagc 
               
               
                   
               
               
                   
                 RN77 
                 tacaggtgccagccaccatgc 
               
               
                   
               
               
                   
                 RN78 
                 gcctgtaatcccagcactctgg 
               
               
                   
               
               
                   
                 RN79 
                 gacagagtcccactcttgttgc 
               
               
                   
               
               
                   
                 RN80 
                 gtgccttccaaagcagtgtagg 
               
               
                   
               
               
                   
                 RN81 
                 tatcttactgggtatgtataatgcc 
               
               
                   
               
               
                   
                 RN82 
                 caaaggaaatacgtcctaccagg 
               
               
                   
               
               
                   
                 RN83 
                 ccttttctcacagacatgcttcc 
               
               
                   
               
               
                   
                 RN84 
                 taaacacagtgagcagaatccc 
               
               
                   
               
               
                   
                 RN85 
                 ataaagcaaacttctaaaagggtcc 
               
               
                   
               
               
                   
                 RN86 
                 accactacactccagcctggg 
               
               
                   
               
               
                   
                 RN87 
                 gatacctgggtcagagtaagtgc 
               
               
                   
               
               
                   
                 RN88 
                 tgtaatctcagctacttgggagg 
               
               
                   
               
               
                   
                 RN89 
                 gtgtcgtcttctcttcctctacg 
               
               
                   
               
               
                   
                 RN90 
                 ctggctagtatgaggttggtgc 
               
               
                   
               
               
                   
                 RN91 
                 ggactagccacatttcaaccagg 
               
               
                   
               
               
                   
                 RN92 
                 gcagtatactgagaatttagtttcc 
               
               
                   
               
               
                   
                 RN93 
                 gaggctgaggcaggagaatgg 
               
               
                   
               
               
                   
                 RN94 
                 cattgtttgatgaaggtcaacagc 
               
               
                   
               
               
                   
                 RN95 
                 cagacaagagtggctacggcag 
               
               
                   
               
               
                   
                 RN96 
                 acgcccagccagattattcagg 
               
               
                   
               
               
                   
                 RN97 
                 ggaaccagaaagaagtgcaaagg 
               
               
                   
               
               
                   
                 RN98 
                 tgagccatcttggaggcaggc 
               
               
                   
               
               
                   
                 RN99 
                 caggaccttcctacaaacctcc 
               
               
                   
               
               
                   
                 RN100 
                 aacacaacatatctgaccttacgc 
               
               
                   
               
               
                   
                 RN101 
                 gccttagaagtccagaggaaagc 
               
               
                   
               
               
                   
                 RN102 
                 tgacgtacccagtagaccttcc 
               
               
                   
               
               
                   
                 RN103 
                 ctctgcaagcctgggaaacagg 
               
               
                   
               
               
                   
                 RN104 
                 gccttgtccccaagtcctaagg 
               
               
                   
               
               
                   
                 RN105 
                 gcaaagggactcctggaattcc 
               
               
                   
               
               
                   
                 RN106 
                 gctcctgcctgtaatcccagc 
               
               
                   
               
               
                   
                 RN107 
                 gaaggaaacagaaaaagcagaggc 
               
               
                   
               
               
                   
                 RN108 
                 cttactaccgttcttcttcactgg 
               
               
                   
               
               
                   
                 RN109 
                 actattctgtttctttaggtttactgc 
               
               
                   
               
               
                   
                 RN110 
                 cggtggctcacacctgtaatcc 
               
               
                   
               
               
                   
                 RN111 
                 agccagagttctgtgctctagg 
               
               
                   
               
               
                   
                 RN112 
                 taatttgcatttcgtgccgctcc 
               
               
                   
               
               
                   
                 RN113 
                 cacttttaatacagatcccaatagg 
               
               
                   
               
               
                   
                 RN114 
                 atgtattttttcttttcctgtcaagc 
               
               
                   
               
               
                   
                 RN115 
                 aaatgttaacattattctccctaagg 
               
               
                   
               
               
                   
                 RN116 
                 catatgcccagatcccgtctcc 
               
               
                   
               
               
                   
                 RN117 
                 acaggtgtgagccgctgcacc 
               
               
                   
               
               
                   
                 RN118 
                 gccaagacgtttacagttttggc 
               
               
                   
               
               
                   
                 RN119 
                 aggaaacttctgaggatgatggg 
               
               
                   
               
               
                   
                 RN120 
                 gctttatagggcagtctgaattcc 
               
               
                   
               
               
                   
                 RN121 
                 ttagaataaaagttatctcgggagg 
               
               
                   
               
               
                   
                 RN122 
                 taatttcttcagctttatccctcag 
               
               
                   
               
               
                   
                 RN123 
                 cacatgactaattctctattcattcc 
               
               
                   
               
               
                   
                 RN124 
                 aaagacctcaagaaaagagtcacc 
               
               
                   
               
               
                   
                 RN125 
                 gacccataaagattatatgcccag 
               
               
                   
               
               
                   
                 RN126 
                 aaagtactaatgcagtgtgtcagc 
               
               
                   
               
               
                   
                 RN127 
                 gaggttcctcgattcccctgc 
               
               
                   
               
               
                   
                 RN128 
                 ggagagcagaggaattcacagg 
               
               
                   
               
               
                   
                 RN129 
                 agtaattagaaactgattctaagacg 
               
               
                   
               
               
                   
                 RN130 
                 cataccattgccaatccagttcc 
               
               
                   
               
               
                   
                 RN131 
                 attacgggtgcctgccactgc 
               
               
                   
               
               
                   
                 RN132 
                 cagccaggcagaggagagagg 
               
               
                   
               
               
                   
                 RN133 
                 ttttcattccaagtttctgtttggg 
               
               
                   
               
               
                   
                 RN134 
                 tttcaaataggaatttggataatccc 
               
               
                   
               
               
                   
                 RN135 
                 taagccgagatcacaccactgc 
               
               
                   
               
               
                   
                 RN136 
                 ccttcagcgcattatatcttggc 
               
               
                   
               
               
                   
                 RN137 
                 ccatctaatccatcttaaattcacc 
               
               
                   
               
               
                   
                 RN138 
                 gagtggagactgcgccactgc 
               
               
                   
               
               
                   
                 RN139 
                 aatcatgtgccaattaaaccatggc 
               
               
                   
               
               
                   
                 RN140 
                 cccagggaccagaccagacc 
               
               
                   
               
               
                   
                 RN141 
                 ctcactcaccagtgaaaatcagc 
               
               
                   
               
               
                   
                 RN142 
                 ggttgctctcgaactcctgacc 
               
               
                   
               
               
                   
                 RN143 
                 gttcccccagctcctttctgc 
               
               
                   
               
               
                   
                 RN144 
                 agaaagatgtagaagggtccagc 
               
               
                   
               
               
                   
                 RN145 
                 gggaaaaggtgtattatgcaagcg 
               
               
                   
               
               
                   
                 RN146 
                 ctctctcagacctaatgcaaaagc 
               
               
                   
               
               
                   
                 RN147 
                 aactatacatacagtatttgtattagc 
               
               
                   
               
               
                   
                 RN148 
                 aaattaatgcaatccatgatccagg 
               
               
                   
               
               
                   
                 RN149 
                 ctttctccactctaagagaaccc 
               
               
                   
               
               
                   
                 RN150 
                 ttttggtgtgttcatattggctgc 
               
               
                   
               
               
                   
                 RN151 
                 gcttccacaaatgacagacaaagg 
               
               
                   
               
               
                   
                 RN152 
                 ggctcatgcttgtaatcccagc 
               
               
                   
               
               
                   
                 RN153 
                 catatgaattgttgttcctttgtagg 
               
               
                   
               
               
                   
                 RN154 
                 cactggtacaagtccaagagtcc 
               
               
                   
               
               
                   
                 RN155 
                 gaccctgtgtctacttcctggg 
               
               
                   
               
               
                   
                 RN156 
                 tatttgaactatctcttgaaatgtcc 
               
               
                   
               
               
                   
                 RN157 
                 ctgattaaaaagtattacccttggc 
               
               
                   
               
               
                   
                 RN158 
                 tttgaaactgcactcaataacttgg 
               
               
                   
               
               
                   
                 RN159 
                 agtaatgtgtcatgatccaatggc 
               
               
                   
               
               
                   
                 RN169 
                 gaaagcatttcccaatgtctcacc 
               
               
                   
               
               
                   
                 RN161 
                 caatggacaaaaggcccaactgc 
               
               
                   
               
               
                   
                 RN162 
                 tccagctctggcttttttgttaag 
               
               
                   
               
               
                   
                 RN163 
                 acggagtctcactccgtgacc 
               
               
                   
               
               
                   
                 RN164 
                 ctatgtcatagtcaagagactttgc 
               
               
                   
               
               
                   
                 RN165 
                 gttcaagcgattctcctgtctcg 
               
               
                   
               
               
                   
                 RN166 
                 ccacctaatacttaaatacggaagc 
               
               
                   
               
               
                   
                 RN167 
                 atattcaacaaacttaatagtgaagtg 
               
               
                   
               
               
                   
                 RN168 
                 ttacaggcgtgagtcaccatgc 
               
               
                   
               
               
                   
                 RN169 
                 aacacctccaagaggccaaacg 
               
               
                   
               
               
                   
                 RN170 
                 tactattggcaaatttcaattatatgg 
               
               
                   
               
               
                   
                 RN171 
                 agcccacatcctaaaattcaataag 
               
               
                   
               
               
                   
                 RN172 
                 gaaagtggataagtgtttgtctgg 
               
               
                   
               
               
                   
                 RN173 
                 ggccaggcattcaagaccagc 
               
               
                   
               
               
                   
                 RN174 
                 agccaacaacaaaaagacacaacc 
               
               
                   
               
               
                   
                 RN175 
                 ttgagcccaggagttcaagacc 
               
               
                   
               
               
                   
                 RN176 
                 cagactaaagatctcagagagaaac 
               
               
                   
               
               
                   
                 RN177 
                 cgcttgtaatcccagcacttgg 
               
               
                   
               
               
                   
                 RN178 
                 aaaagtgaaatcagaatttgtttcc 
               
               
                   
               
               
                   
                 RN179 
                 caggcgtgagcaactgtgtcc 
               
               
                   
               
               
                   
                 RN180 
                 ggtccagtaggatctcgtttgc 
               
               
                   
               
               
                   
                 RN181 
                 actttgaaaatgttgttatagctggg 
               
               
                   
               
               
                   
                 RN182 
                 ttccctgcatctaagtcttctcc 
               
               
                   
               
               
                   
                 RN183 
                 agatatctaccattgaagagtttgc 
               
               
                   
               
               
                   
                 RN184 
                 agtcttcacttcactttgttgtcc 
               
               
                   
               
               
                   
                 RN185 
                 ccatgcaggtatgaaatataaaagc 
               
               
                   
               
               
                   
                 RN186 
                 tgggtgacagagtgagactcc 
               
               
                   
               
               
                   
                 RN187 
                 acagcaataccgggttaacatgc 
               
               
                   
               
               
                   
                 RN188 
                 tttatgtaaaagatgaatgcgaggc 
               
               
                   
               
               
                   
                 RN189 
                 ctactctgctactgggaacagg 
               
               
                   
               
               
                   
                 RN190 
                 caaacgttagtctggcaaaatgcg 
               
               
                   
               
               
                   
                 RN191 
                 tgcacgctaccacacccagc 
               
               
                   
               
               
                   
                 RN192 
                 aattcttggatctgtgtgtttactgc 
               
               
                   
               
               
                   
                 RN193 
                 taccagttatcattctctttctgc 
               
               
                   
               
               
                   
                 RN194 
                 atccacccacctcggcctcc 
               
               
                   
               
               
                   
                 RN195 
                 cactctgcctggcccttaatgg 
               
               
                   
               
               
                   
                 RN196 
                 atagtttgtttaatatgccactaagg 
               
               
                   
               
               
                   
                 RN197 
                 gcgtgagccaccgcacctgg 
               
               
                   
               
               
                   
                 RN198 
                 ctccatcacacaaattttatgtggc 
               
               
                   
               
               
                   
                 RN199 
                 agacggagtctcgttctgtcgc 
               
               
                   
               
               
                   
                 RN200 
                 tcccaggttcaagccattctcc 
               
               
                   
               
               
                   
                 RN201 
                 tattttgagagtctcactctgtcg 
               
               
                   
               
               
                   
                 RN202 
                 gtctcgaactcctgacctcagg 
               
               
                   
               
               
                   
                 RN203 
                 aaggaggtgaagagtgaactacg 
               
               
                   
               
               
                   
                 RN204 
                 gtctcaggttttggacttacttgg 
               
               
                   
               
               
                   
                 RN205 
                 tttacagatcttaaatgcattaggac 
               
               
                   
               
               
                   
                 RN206 
                 gtacactgaacaaaggagacagg 
               
               
                   
               
               
                   
                 RN207 
                 ctggtagtaatgcaaaatagcacc 
               
               
                   
               
               
                   
                 RN208 
                 catttaatgtgaaatgaattataagcc 
               
               
                   
               
               
                   
                 RN209 
                 gagacagggtttcactatgttgg 
               
               
                   
               
               
                   
                 RN210 
                 ccagcactttggaaggctgagg 
               
               
                   
               
               
                   
                 RN211 
                 gaaaccaagtatcatggtaaattgc 
               
               
                   
               
               
                   
                 RN212 
                 cagtgagggctgctcagttcc 
               
               
                   
               
               
                   
                 RN213 
                 gccaggtgcggtggctcacg 
               
               
                   
               
               
                   
                 RN214 
                 catgcctgtaatcccagctacc 
               
               
                   
               
               
                   
                 RN215 
                 atgtaaatggtacagtcactttagg 
               
               
                   
               
               
                   
                 RN216 
                 cccacaatacagagaactcttacc 
               
               
                   
               
               
                   
                 RN217 
                 tgaaacatgcagcccagtgtcc 
               
               
                   
               
               
                   
                 RN218 
                 tgttttttctcctgccttcaatcc 
               
               
                   
               
               
                   
                 RN219 
                 gctttcctgggtctccatctgg 
               
               
                   
               
               
                   
                 RN220 
                 gcagccgcttgaaaacaaaacagc 
               
               
                   
               
               
                   
                 RN221 
                 gatcacgttacatttgggggtgg 
               
               
                   
               
               
                   
                 RN222 
                 taggctgaaaaactaaaatttgttgc 
               
               
                   
               
               
                   
                 RN223 
                 ctcctttgggctcctttagtcc 
               
               
                   
               
               
                   
                 RN224 
                 gcctcggcctcccaaagtgc 
               
               
                   
               
               
                   
                 RN225 
                 aatgcctagagagatttggcagg 
               
               
                   
               
               
                   
                 RN226 
                 gagatggggtttcactatgttgg 
               
               
                   
               
               
                   
                 RN227 
                 tgtgatcttgccactgcactcc 
               
               
                   
               
               
                   
                 RN228 
                 acttctcctccatttgtttcttcg 
               
               
                   
               
               
                   
                 RN229 
                 cgtgcccgggctcagttctac 
               
               
                   
               
               
                   
                 RN230 
                 ccaaaacaataaaatcacaatttggg 
               
               
                   
               
               
                   
                 RN231 
                 ctgaactgccttagagtaaatccg 
               
               
                   
               
               
                   
                 RN232 
                 atttctgtatcaggtctgtgttcc 
               
               
                   
               
               
                   
                 RN233 
                 ggctgaccccttcactgtttcc 
               
               
                   
               
               
                   
                 RN234 
                 caaaaattagccaggcatggtgg 
               
               
                   
               
               
                   
                 RN235 
                 gcagtgagcagtgatcgcacc 
               
               
                   
               
               
                   
                 RN236 
                 aaagactgtgaactaacttgtttgc 
               
               
                   
               
               
                   
                 RN237`1 
                 tgccaagaattacacattattaggc 
               
               
                   
               
               
                   
                 RN238 
                 ggccaggatgtcattaactttcc 
               
               
                   
               
               
                   
                 RN239 
                 gtaagagctgacgtgtattgtgc 
               
               
                   
               
               
                   
                 RN240 
                 cccggtgaggccgcacatcc 
               
               
                   
               
               
                   
                 RN241 
                 cctgcgccttaaccccctcc 
               
               
                   
               
               
                   
                 RN242 
                 cggcgcctaggggccatcg 
               
               
                   
               
               
                   
                 RN243 
                 acttaaggaaacgaacatgacacc 
               
               
                   
               
               
                   
                 RN244 
                 gagaccgagtcttgctgtgtcg 
               
               
                   
               
               
                   
                 RN245 
                 gtattaattgaagatgatttggaatgc 
               
               
                   
               
               
                   
                 RN246 
                 tctttaaaagactatcgctgaggc 
               
               
                   
               
               
                   
                 RN247 
                 aaaagagacatcagtagagcatcc 
               
               
                   
               
               
                   
                 RN248 
                 gttcatgttttctttgacgtctcc 
               
               
                   
               
               
                   
                 RN249 
                 tttcgaaagttcaggctgagtgc 
               
               
                   
               
               
                   
                 RN250 
                 gaccctcaaaacaatcctctaagg 
               
               
                   
               
               
                   
                 RN251 
                 caaaacacacttagaaacaaactgc 
               
               
                   
               
               
                   
                 RN252 
                 gcctgggcgacatagtgagacc 
               
               
                   
               
               
                   
                 RN253 
                 ggcaggagaatggcgtgaacc 
               
               
                   
               
               
                   
                 RN254 
                 tttgctcgttgcccaggctgg 
               
               
                   
               
               
                   
                 RN255 
                 gcaacttaatgtgatagaataatagc 
               
               
                   
               
               
                   
                 RN256 
                 cctccccttctgctgccagc 
               
               
                   
               
               
                   
                 RN257 
                 ccacaacaatgtaaactcctctgg 
               
               
                   
               
               
                   
                 RN258 
                 tactctccctagagttcgttccc 
               
               
                   
               
               
                   
                 RN259 
                 gggtccccctttggccattcc 
               
               
                   
               
               
                   
                 RN260 
                 gatcttggctcacttcaacctcc 
               
               
                   
               
               
                   
                 RN261 
                 aggggaaatatttaaaccttgg 
               
               
                   
               
               
                   
                 RN262 
                 aatgcaatggtgcatttacagagg 
               
               
                   
               
               
                   
                 RN263 
                 tcattttatctatttctacatggtcc 
               
               
                   
               
               
                   
                 RN264 
                 ggaagggaaatgcccatgaacc 
               
               
                   
               
               
                   
                 RN265 
                 agtgaacattttctgcagcctcc 
               
               
                   
               
               
                   
                 RN266 
                 caacaggacgtcaggcgatcc 
               
               
                   
               
               
                   
                 RN267 
                 ccttcaggctgtcctgaaaagg 
               
               
                   
               
               
                   
                 RN268 
                 agtctcactccatcgcccagg 
               
               
                   
               
               
                   
                 RN269 
                 actgtgaacagtagttaactcagg 
               
               
                   
               
               
                   
                 RN270 
                 gcatgcctgtaatccaagctgc 
               
               
                   
               
               
                   
                 RN271 
                 gaaacaattctcttttcacacttgc 
               
               
                   
               
               
                   
                 RN272 
                 ggctcatgcctgttatcccagc 
               
               
                   
               
               
                   
                 RN273 
                 agaagaagcttagtcatatgtttgg 
               
               
                   
               
               
                   
                 RN274 
                 cagatgcttgagccaaacaaatgg 
               
               
                   
               
               
                   
                 RN275 
                 ctggcagacagagtgagactcc 
               
               
                   
               
               
                   
                 RN276 
                 aatgtgtgaatattattcattacaggg 
               
               
                   
               
               
                   
                 RN277 
                 gcaggagaattgcttgaacctgg 
               
               
                   
               
               
                   
                 RN278 
                 ctttagtcaaattaaaacagtctatcc 
               
               
                   
               
               
                   
                 RN279 
                 gatttctatctcctgcaaccacc 
               
               
                   
               
               
                   
                 RN280 
                 ttcttgtgtaactactaaaaatctcc 
               
               
                   
               
               
                   
                 RN281 
                 aaagggtcttcataaggctaatgg 
               
               
                   
               
               
                   
                 RN282 
                 ctcttaaggattatttatatgaagacc 
               
             
          
         
       
     
     Example 3 
     Identification of the PML-RARalpha Breakpoint 
     Amplified patient DNA was electrophoresed on a 2% agarose gel. P is patient DNA, N is the normal DNA and W is the water control. The patient DNA was amplified using multiple RARα primers and a single PML primer
         a) Amplified patient DNA electrophoresed on a 2% agarose gel, P is patient DNA, N is the normal DNA and W is the water control. The patient DNA was amplified for one round using an RARα primer and a PML primer designed using the breakpoint sequence.   b) The sequence chromatogram obtained from the patient DNA. The breakpoint between PML and RARα is shown.
 
Isolation of the PML-RARalpha Breakpoint in Acute Promyelocytic Leukemia
       

     Two patients have been studied and the breakpoint has been isolated and sequenced in both. The primers used are shown in Example 4. 
     Example 4 
     Primers Used for Isolation of PML-RARalpha Translocation Breakpoint in Acute Promyelocytic Leukemia 
     PML Forward Primers 
     
       
         
               
               
               
             
           
               
                   
                 1st Rd 
                   
               
               
                   
                 PML F1-FT1 
                 caggaggagccccagagc 
               
               
                   
               
               
                   
                 PML F2-FT1 
                 tcctggggatggttggatgc 
               
               
                   
               
               
                   
                 PML F3-FT1 
                 tgaccccacagagtttacacagc 
               
               
                   
               
               
                   
                 PML F4-FT1 
                 agtcagggcaggctctgcc 
               
               
                   
               
               
                   
                 PML F5-FT1 
                 tattttggccccatccagaaagc 
               
               
                   
               
               
                   
                 PML F6-FT1 
                 cacccagagtacagctttgttcc 
               
               
                   
               
               
                   
                 2nd Rd 
                   
               
               
                   
                 PML F1-FT2 
                 gaggagccccagagcctgc 
               
               
                   
               
               
                   
                 PML F2-FT2 
                 tggggatggttggatgcttacc 
               
               
                   
               
               
                   
                 PML F3-FT2 
                 cccacagagtttacacagcttgc 
               
               
                   
               
               
                   
                 PML F4-FT2 
                 caggctctgcccactcacc 
               
               
                   
               
               
                   
                 PML F5-FT2 
                 ccatccagaaagcccaaagcc 
               
               
                   
               
               
                   
                 PML F6-FT2 
                 ccagagtacagctttgttcctcattc 
               
             
          
         
       
     
     The second round primers were internal to the first round primers and were used for performing Bottleneck PCR in order to eliminate non-specific amplified material and facilitate isolation of the translocation breakpoint. 
     Various combinations of the forward and reverse primers can be used. 2 exemplary protocols were either to set up 6 PCRs, each containing a different PML primer and all 34 RARalpha primers, or to set up 1 PCR which contained all 6 forward and all 34 reverse primers. 
     34 Reverse RARalpha Reverse Primers Used for the First PCR Round and the Tag Sequence which was on the 5′ End of Each Primer 
     
       
         
               
               
               
             
           
               
                   
                 Tag R1 
                 gcagtacaaacaacgcacagcg 
               
               
                   
               
               
                   
                 RAR1 
                 ctgccaccctccacagtccc 
               
               
                   
               
               
                   
                 RAR2 
                 gccaagaccatgcatgcg 
               
               
                   
               
               
                   
                 RAR3 
                 cccagggacaaagagactccc 
               
               
                   
               
               
                   
                 RAR4 
                 caggaagcagacagtcttctagttcc 
               
               
                   
               
               
                   
                 RAR5 
                 tgcctgtaatcccaacactttgg 
               
               
                   
               
               
                   
                 RAR6 
                 tccctctggccaggatggg 
               
               
                   
               
               
                   
                 RAR7 
                 atggggaatgggagtaggaagc 
               
               
                   
               
               
                   
                 RAR8 
                 cagatcagttctcccctccagc 
               
               
                   
               
               
                   
                 RAR9 
                 acaaaaaagaaacatgctcagagagg 
               
               
                   
               
               
                   
                 RAR10 
                 tggtggcatgcatctgtagtcc 
               
               
                   
               
               
                   
                 RAR11 
                 aggtgctctatagatgttagcatccc 
               
               
                   
               
               
                   
                 RAR12 
                 ccaggacaggatggagatctgg 
               
               
                   
               
               
                   
                 RAR13 
                 agggaacctgtgcattatccttgc 
               
               
                   
               
               
                   
                 RAR14 
                 cagaagtcttgctttaaggaggagg 
               
               
                   
               
               
                   
                 RAR15 
                 gggtacgtgaaactcaccaagg 
               
               
                   
               
               
                   
                 RAR16 
                 cagagtgtggcaagcaaggg 
               
               
                   
               
               
                   
                 RAR17 
                 aacattttaaaggtacaaataacgtggg 
               
               
                   
               
               
                   
                 RAR18 
                 tagggagcaacagccattaagc 
               
               
                   
               
               
                   
                 RAR19 
                 ggtgcactgtccagctctgg 
               
               
                   
               
               
                   
                 RAR20 
                 actctcgctgaactcgcctgg 
               
               
                   
               
               
                   
                 RAR21 
                 ctcggtctctggtggtacgc 
               
               
                   
               
               
                   
                 RAR22 
                 gcaagaggtccgagctggg 
               
               
                   
               
               
                   
                 RAR23 
                 ggaagaagtgaaacaagagatgaagg 
               
               
                   
               
               
                   
                 RAR24 
                 cccagagaacaaaccggattagg 
               
               
                   
               
               
                   
                 RAR25 
                 cccttcaaccttctccaatctgc 
               
               
                   
               
               
                   
                 RAR26 
                 cccatgtccagtggtttaggg 
               
               
                   
               
               
                   
                 RAR27 
                 gagattggtgggagacagatgg 
               
               
                   
               
               
                   
                 RAR28 
                 cttctcagctcaaagttccagcg 
               
               
                   
               
               
                   
                 RAR29 
                 gaatgggagagatgaccagagg 
               
               
                   
               
               
                   
                 RAR30 
                 aagggcaagggggtatgtgg 
               
               
                   
               
               
                   
                 RAR31 
                 ggaaggaagcatgggaacacc 
               
               
                   
               
               
                   
                 RAR32 
                 ccatcaatgctctgtctgtctgg 
               
               
                   
               
               
                   
                 RAR33 
                 gtgccgtgactgtgcttgg 
               
               
                   
               
               
                   
                 RAR34 
                 acatcccattgacctcatcaagc 
               
             
          
         
       
     
     Nearly all translocations involve a 3 kb region of the BCR gene and 140 kb region of the ABL gene. Six forward primers used to cover the region of the BCR gene and 282 primers used to cover the region of the ABL gene. Six PCRs are set up, each containing one of the BCR primers, all of the ABL primers, and the common tag primer. 
     If necessary, a second round of PCR is performed with a nested internal BCR primer and 282 nested internal ABL primers Alternatively, 1-3 rounds of Bottleneck PCR are performed in order to remove non-specific amplified products and reveal the amplified translocation sequence. 
     The ABL gene is very rich in Alu sequences, and the BCR gene also contains one such sequence. The ABL primers have therefore undergone a selection procedure which sequentially involves, for each ABL primer:
         design using standard criteria   pairing with each BCR primer and testing by electronic PCR for amplification off the BCR template. Primers that fail this criterion are discarded.   incorporation in a pool of 12 or 24 ABL primers, pairing the pool with each BCR primer, and testing by experimental PCR using a BCR template which has been previously produced by PCR amplification. Any pool that that produces amplification and thus fails this test is further analysed by testing each of the individual ABL primers to determine which is responsible for amplification. When identified, this primer is discarded.       

     The BCR and ABL primers used in Example 1 are shown in Example 2. 
     Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. 
     
       
         
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Sequence Identifier 
                 Sequence 
               
               
                   
               
             
             
               
                 SEQ ID NO:001 
                 gtgggcccccccgtttccgtgtacagggcac 
               
               
                   
                 ctgcagggagggcaggcagctagcctgaagg 
               
               
                   
                 ctgatccccccttcctgttagcacttttgat 
               
               
                   
                 gggactagtggactttggttcagaaggaaga 
               
               
                   
                 gctatgcttgttagggcctcttgtctcctcc 
               
               
                   
                 caggagtggacaaggtgggttaggagcagtt 
               
               
                   
                 tctccctgagtggctgc 
               
               
                   
               
               
                 SEQ ID NO:002 
                 caccacgtctggctaatttttgtatttttag 
               
               
                   
                 tagagatggggtttcaacatgttagccaggc 
               
               
                   
                 tggtctcgaactcctgacctcaggtgatcca 
               
               
                   
                 cccgcctgggccctccaaagtgctgggatta 
               
               
                   
                 caggcaggagccactgtgcccggcctgacct 
               
               
                   
                 catatttgaataccgagttttagttctggag 
               
               
                   
                 gagctgcaggttttatgaaaagggaacacat 
               
               
                   
                 ttgattcctcagagcagccacaggccagctc 
               
               
                   
                 tctgaagtaaagtgcacgtgtgcatgtgtgt 
               
               
                   
                 gcacactcacacacacgtacacacacattca 
               
               
                   
                 caaataactgtgcccggcctgacctcatatt 
               
               
                   
                 tgaataccgagttttagttctggaggagctg 
               
               
                   
                 cagg 
               
               
                   
               
               
                 SEQ ID NO:003 
                 tttgggaggctgaggcaggtggatcgcttga 
               
               
                   
                 gctcaggagttggagaccagcctgaccaaca 
               
               
                   
                 tggtgaaaccctgtgtctactaaaaatacaa 
               
               
                   
                 agattagccgggctaggcagtgggcacctgt 
               
               
                   
                 aatcacaactgcttgggaggctgagggaaga 
               
               
                   
                 gaatcgcttgaacccaggaggcggaggttgc 
               
               
                   
                 agtgagccgagcttgtgccactgcattccag 
               
               
                   
                 cctgggcgacagag 
               
               
                   
               
               
                 SEQ ID NO:004 
                 ggtctcactctgttgaactcctggtggcctc 
               
               
                   
                 aagggatcctcctacctcggcctcacaaagt 
               
               
                   
                 attggaattacaggtgtgagtcactgcagct 
               
               
                   
                 ggccttcacttatcactgtgaggagtaaaca 
               
               
                   
                 gctgcatggtgggcttaatgccatctaacac 
               
               
                   
                 gagtgactccatgttcagacagtaggatcac 
               
               
                   
                 aaatgattattatatagcaatgaatggccac 
               
               
                   
                 aggtacatagactaaggagccacatccctgc 
               
               
                   
                 t 
               
               
                   
               
               
                 SEQ ID NO:005 
                 cctccagctacctgccagccggcacttttgg 
               
               
                   
                 tcaagctgttttgcattcactgttgcacata 
               
               
                   
                 tgctcagtcacacacacagcatacgctatgc 
               
               
                   
                 acatgtgtccacacacaccccacccacatcc 
               
               
                   
                 cacatcaccccgaccccctctgctgtccttg 
               
               
                   
                 gaaccttattacacttcgagtcactggtttg 
               
               
                   
                 cctgtattgtgaaaccagctggatcc 
               
               
                   
               
               
                 SEQ ID NO:006 
                 ttatttataacaacattttcagcgtggcaac 
               
               
                   
                 tgcagtttcagaatggtggaattataccagt 
               
               
                   
                 cagagagagatgcaaatgatttaaaatagga 
               
               
                   
                 agaaagcaggtgtctggcccagaggaccaga 
               
               
                   
                 ttaagaagaccccatgagagttacaatagtt 
               
               
                   
                 agtgaaaatggtgcttctgcaaacctcatgt 
               
               
                   
                 ctacagaagctggt 
               
               
                   
               
               
                 SEQ ID NO:007 
                 tgcaccttcataacataatctttctcctggg 
               
               
                   
                 cccctgtctctggctgcctcataaacgctgg 
               
               
                   
                 tgtttccctcgtgggcctccctgcatccctg 
               
               
                   
                 catctcctcccgggtcctgtctgtgagcaat 
               
               
                   
                 acagcgtgacaccctacgctgccccgtggtc 
               
               
                   
                 ccgggcttgtctctccttgcctccctgttac 
               
               
                   
                 ctttctttctatctcttccttgccccg 
               
               
                   
               
               
                 SEQ ID NO:008 
                 gtgagctccgcctcctgtcagatcagtggcg 
               
               
                   
                 gcattagtttctcataggagcatgaaatcta 
               
               
                   
                 ttgtgaacagtacatgcgatggatccaggtt 
               
               
                   
                 gcgtgctcctagtgagaatctaatgcctgag 
               
               
                   
                 gatctctcattgtctcttatcactcccagat 
               
               
                   
                 aggactgtctagttgcaggaaaacaagctca 
               
               
                   
                 gggctcccactgattctacattacagtgggt 
               
               
                   
                 tgtataattattatatattacaatgtaataa 
               
               
                   
                 taa 
               
               
                   
               
               
                 SEQ ID NO:009 
                 ggagtctgaggaggggaaggaggcaaggttg 
               
               
                   
                 gctcggatcccagccagtaagtctgggtgtg 
               
               
                   
                 g 
               
               
                   
               
               
                 SEQ ID NO:010 
                 cttctccctgacatccgtgg 
               
               
                   
               
               
                 SEQ ID NO:011 
                 acacagcatacgctatgcacatgtg 
               
               
                   
               
               
                 SEQ ID NO:012 
                 gaggttgttcagatgaccacgg 
               
               
                   
               
               
                 SEQ ID NO:013 
                 cagctactggagctgtcagaacag 
               
               
                   
               
               
                 SEQ ID NO:014 
                 tgggcctccctgcatcc 
               
               
                   
               
               
                 SEQ ID NO:015 
                 tccccctgcaccccacg 
               
               
                   
               
               
                 SEQ ID NO:016 
                 tgacatccgtggagctgcagatgc 
               
               
                   
               
               
                 SEQ ID NO:017 
                 acatgtgtccacacacaccccacc 
               
               
                   
               
               
                 SEQ ID NO:018 
                 accacgggacacctttgaccctgg 
               
               
                   
               
               
                 SEQ ID NO:019 
                 ctggagctgtcagaacagtgaagg 
               
               
                   
               
               
                 SEQ ID NO:020 
                 tccctgcatccctgcatctcctcc 
               
               
                   
               
               
                 SEQ ID NO:021 
                 cccacgacttctccagcactgagc 
               
               
                   
               
               
                 SEQ ID NO:022 
                 gcaacactgtgacgtactggagg 
               
               
                   
               
               
                 SEQ ID NO:023 
                 gtctatctaaaattcacaaggaatgc 
               
               
                   
               
               
                 SEQ ID NO:024 
                 aggcaaagtaaaatccaagcaccc 
               
               
                   
               
               
                 SEQ ID NO:025 
                 cactcctgcactccagcctgg 
               
               
                   
               
               
                 SEQ ID NO:026 
                 caaccaccaaagtgcttttcctgg 
               
               
                   
               
               
                 SEQ ID NO:027 
                 atatggcatctgtaaatattaccacc 
               
               
                   
               
               
                 SEQ ID NO:028 
                 tgcctcggcctcccaaagtgc 
               
               
                   
               
               
                 SEQ ID NO:029 
                 agccaccacacccagccagg 
               
               
                   
               
               
                 SEQ ID NO:030 
                 aataactgttttctccccccaaaac 
               
               
                   
               
               
                 SEQ ID NO:031 
                 tgttttacaaaaatggggccatacc 
               
               
                   
               
               
                 SEQ ID NO:032 
                 acttaagcaaattctttcataaaaaggg 
               
               
                   
               
               
                 SEQ ID NO:033 
                 ctttcaattgttgtaccaactctcc 
               
               
                   
               
               
                 SEQ ID NO:034 
                 acctcctgcatctctccttttgc 
               
               
                   
               
               
                 SEQ ID NO:035 
                 aaataaagttttgagaaccataagtgg 
               
               
                   
               
               
                 SEQ ID NO:036 
                 caccatcacagctcactgcagc 
               
               
                   
               
               
                 SEQ ID NO:037 
                 aacctctttgagaatcggatagcc 
               
               
                   
               
               
                 SEQ ID NO:038 
                 aaataaagtacatacctccaattttgc 
               
               
                   
               
               
                 SEQ ID NO:039 
                 gacacattcctatgggtttaattcc 
               
               
                   
               
               
                 SEQ ID NO:040 
                 tgtaaaatatggtttcagaagggagg 
               
               
                   
               
               
                 SEQ ID NO:041 
                 gcaggtggataacgaggtcagg 
               
               
                   
               
               
                 SEQ ID NO:042 
                 ccagccaagaatttcaaagattagc 
               
               
                   
               
               
                 SEQ ID NO:043 
                 gaagggagatgacaaagggaacg 
               
               
                   
               
               
                 SEQ ID NO:044 
                 gcagaagaactgcttgaacctgg 
               
               
                   
               
               
                 SEQ ID NO:045 
                 gtggtcccagctactcgagagg 
               
               
                   
               
               
                 SEQ ID NO:046 
                 ccctcagcaaaactaactgaaaagg 
               
               
                   
               
               
                 SEQ ID NO:047 
                 tagaaaccaagatatctagaattccc 
               
               
                   
               
               
                 SEQ ID NO:048 
                 ccacgcccggcggaataaatgc 
               
               
                   
               
               
                 SEQ ID NO:049 
                 acaaaaaaagaggcaaaaactgagag 
               
               
                   
               
               
                 SEQ ID NO:050 
                 ctgggcgcagtggctcatgcc 
               
               
                   
               
               
                 SEQ ID NO:051 
                 tggctgtgaggctgagaactgc 
               
               
                   
               
               
                 SEQ ID NO:052 
                 ctgggcgacagagtgagactcc 
               
               
                   
               
               
                 SEQ ID NO:053 
                 aagtctggctgggcgcagtgg 
               
               
                   
               
               
                 SEQ ID NO:054 
                 aatggacaaaagaggtgaactggc 
               
               
                   
               
               
                 SEQ ID NO:055 
                 gatagagtgaaaacgcacaatggc 
               
               
                   
               
               
                 SEQ ID NO:056 
                 aattaaacagctaggtcaatatgagg 
               
               
                   
               
               
                 SEQ ID NO:057 
                 ggtctccactatcaagggacaag 
               
               
                   
               
               
                 SEQ ID NO:058 
                 aagcagctgttagtcatttccagg 
               
               
                   
               
               
                 SEQ ID NO:059 
                 aggcatcctcagattatggctcc 
               
               
                   
               
               
                 SEQ ID NO:060 
                 cctgagtaacactgagaccctgc 
               
               
                   
               
               
                 SEQ ID NO:061 
                 aacactcaagctgtcaagagacac 
               
               
                   
               
               
                 SEQ ID NO:062 
                 attcaggccaggcgcagtggc 
               
               
                   
               
               
                 SEQ ID NO:063 
                 taaatcgtaaaactgccacaaagc 
               
               
                   
               
               
                 SEQ ID NO:064 
                 cagaggagtaggagaaggaaaagg 
               
               
                   
               
               
                 SEQ ID NO:065 
                 ggtagctatctaccaagtagaatcc 
               
               
                   
               
               
                 SEQ ID NO:066 
                 atcagattggaaaaagtcccaaagc 
               
               
                   
               
               
                 SEQ ID NO:067 
                 ctcctgaaaagcacctactcagc 
               
               
                   
               
               
                 SEQ ID NO:068 
                 ctccttaaacctgaggtactggg 
               
               
                   
               
               
                 SEQ ID NO:069 
                 ttttctcctaatagaccaccattcc 
               
               
                   
               
               
                 SEQ ID NO:070 
                 ctgctgtattaccatcactcatgtc 
               
               
                   
               
               
                 SEQ ID NO:071 
                 ctggccaacatagtgaaaccacg 
               
               
                   
               
               
                 SEQ ID NO:072 
                 atttgaataggggttaaagtatcattg 
               
               
                   
               
               
                 SEQ ID NO:073 
                 cacttcagtggaagttggcatgc 
               
               
                   
               
               
                 SEQ ID N 0:074 
                 gtttttcttcgaagtgataaacatacg 
               
               
                   
               
               
                 SEQ ID NO:075 
                 gctccttagtctatgtacctgtgg 
               
               
                   
               
               
                 SEQ ID NO:076 
                 tactctggcatggtaactggtgc 
               
               
                   
               
               
                 SEQ ID NO:077 
                 acaaaggactaggtctgtggagc 
               
               
                   
               
               
                 SEQ ID NO:078 
                 ccaagtttaccaaattaccaaagttacc 
               
               
                   
               
               
                 SEQ ID NO:079 
                 tgagccgatatcacgccactgc 
               
               
                   
               
               
                 SEQ ID NO:080 
                 tcccaataaaggttttggcccagg 
               
               
                   
               
               
                 SEQ ID NO:081 
                 ctgggtagcaaattagggaacagg 
               
               
                   
               
               
                 SEQ ID NO:082 
                 ctggccagaaaagacagttttatcc 
               
               
                   
               
               
                 SEQ ID NO:083 
                 ggttcccaggaagggataacacc 
               
               
                   
               
               
                 SEQ ID NO:084 
                 tcactccaggaggttccatttcc 
               
               
                   
               
               
                 SEQ ID NO:085 
                 aggcttggaaataagcagcagtgg 
               
               
                   
               
               
                 SEQ ID NO:086 
                 attcatacaatggaatactactcagc 
               
               
                   
               
               
                 SEQ ID NO:087 
                 taagtgatcctcccacctcaacc 
               
               
                   
               
               
                 SEQ ID NO:088 
                 tataagaggaagactggggctgg 
               
               
                   
               
               
                 SEQ ID NO:089 
                 tcatacttatgcaggttataggagg 
               
               
                   
               
               
                 SEQ ID NO:090 
                 caagatcacgccactgcactcc 
               
               
                   
               
               
                 SEQ ID NO:091 
                 aaaataaatagctggtgctcaagatc 
               
               
                   
               
               
                 SEQ ID NO:092 
                 caccagcctcattcaacagatgg 
               
               
                   
               
               
                 SEQ ID NO:093 
                 caatgcagcctcaacctcctgg 
               
               
                   
               
               
                 SEQ ID NO:094 
                 gttaggtcaggtgctcatgtctg 
               
               
                   
               
               
                 SEQ ID NO:095 
                 aagtttcaaaaggacatgtacaaaatg 
               
               
                   
               
               
                 SEQ ID NO:096 
                 tcctgaagaggctgcagcttcc 
               
               
                   
               
               
                 SEQ ID NO:097 
                 ctggtgcacattcccaagtgtgc 
               
               
                   
               
               
                 SEQ ID NO:098 
                 catgttggccatgttcttctgagg 
               
               
                   
               
               
                 SEQ ID NO:09 
                 9 ctcagcctcccgagtagctgg 
               
               
                   
               
               
                 SEQ ID NO:100 
                 aaagacatttaagaggagatgaggc 
               
               
                   
               
               
                 SEQ ID NO:101 
                 tgctgggattacaggcgtgagc 
               
               
                   
               
               
                 SEQ ID NO:102 
                 tgtgacttccatccgcagctcc 
               
               
                   
               
               
                 SEQ ID NO:103 
                 gacacttttgtggagctttcatgg 
               
               
                   
               
               
                 SEQ ID NO:104 
                 catgtgagggggcacgtcttgc 
               
               
                   
               
               
                 SEQ ID NO:105 
                 tcttctctatgagaaaagtggttgc 
               
               
                   
               
               
                 SEQ ID NO:106 
                 tggcaaaatgctatcgagctgcc 
               
               
                   
               
               
                 SEQ ID NO:107 
                 tatgaacacagccggcctcagg 
               
               
                   
               
               
                 SEQ ID NO:108 
                 gaggttgcagtgagctgagatcg 
               
               
                   
               
               
                 SEQ ID NO:109 
                 gtcaagcacccagtccgatacc 
               
               
                   
               
               
                 SEQ ID NO:110 
                 atctgggcttggtggcgcacg 
               
               
                   
               
               
                 SEQ ID NO:111 
                 gttaagcgggtcccacatcagc 
               
               
                   
               
               
                 SEQ ID NO:112 
                 cagccagtttcagtagaaagatgc 
               
               
                   
               
               
                 SEQ ID NO:113 
                 gacccaagcataaggggactagc 
               
               
                   
               
               
                 SEQ ID NO:114 
                 cccaaaaagtttacaagagaaattttc 
               
               
                   
               
               
                 SEQ ID NO:115 
                 cgcctgtagtcccagctactcg 
               
               
                   
               
               
                 SEQ ID NO:116 
                 cgcgtgatgcggaaaagaaatcc 
               
               
                   
               
               
                 SEQ ID NO:117 
                 tctactatgaaccctccttcagac 
               
               
                   
               
               
                 SEQ ID NO:118 
                 gtgctgggattacaggtgtgagc 
               
               
                   
               
               
                 SEQ ID NO:119 
                 ttatccaaatgtcccagggcagg 
               
               
                   
               
               
                 SEQ ID NO:120 
                 ctgccagcactgctcgccagc 
               
               
                   
               
               
                 SEQ ID NO:121 
                 gctactgcaggcagtgccttcc 
               
               
                   
               
               
                 SEQ ID NO:122 
                 catccaagcccaaggtgtcagg 
               
               
                   
               
               
                 SEQ ID NO:123 
                 tgtttgcatgtaatttcaggaagcc 
               
               
                   
               
               
                 SEQ ID NO:124 
                 gatccgtcactgttaacactcagg 
               
               
                   
               
               
                 SEQ ID NO:125 
                 ctcacagtcacaagctcctgagc 
               
               
                   
               
               
                 SEQ ID NO:126 
                 gagatgatgctggggtcacagg 
               
               
                   
               
               
                 SEQ ID NO:127 
                 ttagaagaatgggatcgcaaagg 
               
               
                   
               
               
                 SEQ ID NO:128 
                 cggtattcaaatatgaggtcaggc 
               
               
                   
               
               
                 SEQ ID NO:129 
                 gtaaatcctgctgccagtcttcc 
               
               
                   
               
               
                 SEQ ID NO:130 
                 acagggtcagacagagccttgg 
               
               
                   
               
               
                 SEQ ID NO:131 
                 agttattgatctaactatacaacaagc 
               
               
                   
               
               
                 SEQ ID NO:132 
                 aaagactaggggccggggacg 
               
               
                   
               
               
                 SEQ ID NO:133 
                 ctggtagaaataaagacaacaaagcc 
               
               
                   
               
               
                 SEQ ID NO:134 
                 gtgccaagtaattaaaagtttgaaacc 
               
               
                   
               
               
                 SEQ ID NO:135 
                 ggcttttgaagggagcaccacc 
               
               
                   
               
               
                 SEQ ID NO:136 
                 gaaggataaatacctatgatactttcc 
               
               
                   
               
               
                 SEQ ID NO:137 
                 ggcagggaaatactgtgcttcaag 
               
               
                   
               
               
                 SEQ ID NO:138 
                 gtggtgaaattccacctcagtacc 
               
               
                   
               
               
                 SEQ ID NO:139 
                 tcccaaagtgctgggattacagg 
               
               
                   
               
               
                 SEQ ID NO:140 
                 gaaattagcaaacaatgccaagacg 
               
               
                   
               
               
                 SEQ ID NO:141 
                 taagtattggaccgggaaggagg 
               
               
                   
               
               
                 SEQ ID NO:142 
                 ctatcattttgctcaaagtgtagcc 
               
               
                   
               
               
                 SEQ ID NO:143 
                 atttcacaaactacagaggccagg 
               
               
                   
               
               
                 SEQ ID NO:144 
                 tagacttctgtctctctatgctgc 
               
               
                   
               
               
                 SEQ ID NO:145 
                 tgagtgagctgccatgtgatacc 
               
               
                   
               
               
                 SEQ ID NO:146 
                 acttcacaccagcctgtccacc 
               
               
                   
               
               
                 SEQ ID NO:147 
                 taactcatatcctcagagagaccc 
               
               
                   
               
               
                 SEQ ID NO:148 
                 agaggttcctcgattcccctgc 
               
               
                   
               
               
                 SEQ ID NO:149 
                 gtgtcagcgtcccaacacaaagc 
               
               
                   
               
               
                 SEQ ID NO:150 
                 gaaagtggatgggcaagcattgc 
               
               
                   
               
               
                 SEQ ID NO:151 
                 gtgatcacctcacagctgcagg 
               
               
                   
               
               
                 SEQ ID NO:152 
                 gtttgtttagtcaaggcatttcacc 
               
               
                   
               
               
                 SEQ ID NO:153 
                 cctcagcctccagagtagctgg 
               
               
                   
               
               
                 SEQ ID NO:154 
                 taaaagaaaactcctccttcctgg 
               
               
                   
               
               
                 SEQ ID NO:155 
                 aatgtgctatgtctttaaatccatgg 
               
               
                   
               
               
                 SEQ ID NO:156 
                 agctggcaaatctggtaatataaaag 
               
               
                   
               
               
                 SEQ ID NO:157 
                 gcttgaacctggaaggtggagg 
               
               
                   
               
               
                 SEQ ID NO:158 
                 gcaggcatgctaagaccttcagc 
               
               
                   
               
               
                 SEQ ID NO:159 
                 cagctccatgaataactccacagg 
               
               
                   
               
               
                 SEQ ID NO:160 
                 gcttgaacccaggaggcagagg 
               
               
                   
               
               
                 SEQ ID NO:161 
                 atcgaagatgccactgcaagagg 
               
               
                   
               
               
                 SEQ ID NO:162 
                 ccaaccacacttcaggggatacc 
               
               
                   
               
               
                 SEQ ID NO:163 
                 cacgccagtccactgatactcac 
               
               
                   
               
               
                 SEQ ID NO:164 
                 gggtttcaccatgttggccagg 
               
               
                   
               
               
                 SEQ ID NO:165 
                 cccaacaaaggctctggcctgg 
               
               
                   
               
               
                 SEQ ID NO:166 
                 atgacagcagaggagcttcatcc 
               
               
                   
               
               
                 SEQ ID NO:167 
                 gcaggctacgagtaaaaggatgg 
               
               
                   
               
               
                 SEQ ID NO:168 
                 cgggtaaaatcttgcctccttcc 
               
               
                   
               
               
                 SEQ ID NO:169 
                 aaacttaaaccaatggtggatgtgg 
               
               
                   
               
               
                 SEQ ID NO:170 
                 agagactgaggaactgttccagc 
               
               
                   
               
               
                 SEQ ID NO:171 
                 gaaacggtcttggatcactgatcc 
               
               
                   
               
               
                 SEQ ID NO:172 
                 tgcgcatgatatcttgtttcaggg 
               
               
                   
               
               
                 SEQ ID NO:173 
                 ggcctccgtttaaactgttgtgc 
               
               
                   
               
               
                 SEQ ID NO:174 
                 gaatgctggcccgacacagtgg 
               
               
                   
               
               
                 SEQ ID NO:175 
                 tcttggtatagaaaagccagctgg 
               
               
                   
               
               
                 SEQ ID NO:176 
                 gcaaaagcccaagagcccctgg 
               
               
                   
               
               
                 SEQ ID NO:177 
                 ttctcccaaaatgagccccaagg 
               
               
                   
               
               
                 SEQ ID NO:178 
                 gtggtgacgtaaacaaaaggtacc 
               
               
                   
               
               
                 SEQ ID NO:179 
                 gcaaattccatgtgaatcttattggc 
               
               
                   
               
               
                 SEQ ID NO:180 
                 cctgatctatggaacagtggtgg 
               
               
                   
               
               
                 SEQ ID NO:181 
                 gttacaaacgttgcagtttgcaacg 
               
               
                   
               
               
                 SEQ ID NO:182 
                 gaaccccgtcaacagtgatcacc 
               
               
                   
               
               
                 SEQ ID NO:183 
                 acaggacctcaaggcaaggagc 
               
               
                   
               
               
                 SEQ ID NO:184 
                 catacctaaaatagaaatgtctatccc 
               
               
                   
               
               
                 SEQ ID NO:185 
                 gagttgcatatatgttttataaatccc 
               
               
                   
               
               
                 SEQ ID NO:186 
                 tgagcccacatccataaagttagc 
               
               
                   
               
               
                 SEQ ID NO:187 
                 accgcaacctttgccgcctgg 
               
               
                   
               
               
                 SEQ ID NO:188 
                 taaatattttgtatggagtcaccacc 
               
               
                   
               
               
                 SEQ ID NO:189 
                 aaagccaggagaaaaagttatgagg 
               
               
                   
               
               
                 SEQ ID NO:190 
                 tcccaaagtcccaggattacagg 
               
               
                   
               
               
                 SEQ iD NO:191 
                 tcactatggagcatctccgatgg 
               
               
                   
               
               
                 SEQ ID NO:192 
                 agttccctggaagtctccgagg 
               
               
                   
               
               
                 SEQ ID NO:193 
                 aaaataatcacccagcccacatcc 
               
               
                   
               
               
                 SEQ ID NO:194 
                 acaaaactacagacacagaaagtgg 
               
               
                   
               
               
                 SEQ ID NO:195 
                 tttgggaggctgaggtaggtgg 
               
               
                   
               
               
                 SEQ ID NO:196 
                 aaagacagtgaaacatctataaggg 
               
               
                   
               
               
                 SEQ ID NO:197 
                 cattttgggagaccagggcagg 
               
               
                   
               
               
                 SEQ ID NO:198 
                 gcatgggacagacacaaagcagc 
               
               
                   
               
               
                 SEQ ID NO:199 
                 gaataacaaagagagccggctgg 
               
               
                   
               
               
                 SEQ ID NO:200 
                 taaaccttttattgaaaattgtcaaatgg 
               
               
                   
               
               
                 SEQ ID NO:201 
                 cgcctcagcctcccaaagtgc 
               
               
                   
               
               
                 SEQ ID NO:202 
                 tacattagttttataggtccagtagg 
               
               
                   
               
               
                 SEQ ID NO:203 
                 gaaggtttattcatattaaaatgtgcc 
               
               
                   
               
               
                 SEQ ID NO:204 
                 ctggcttctgtggtttgagttgg 
               
               
                   
               
               
                 SEQ ID NO:205 
                 acagacctacctcctaaggatgg 
               
               
                   
               
               
                 SEQ ID NO:206 
                 gctagcttttgtgtgtaagaatggg 
               
               
                   
               
               
                 SEQ ID NO:207 
                 ggcctactcacacaatagaatacc 
               
               
                   
               
               
                 SEQ ID NO:208 
                 gcaccattgcactccagcctgg 
               
               
                   
               
               
                 SEQ ID NO:209 
                 gaaattaggataaaggttgtcacagc 
               
               
                   
               
               
                 SEQ ID NO:210 
                 cagaagtgttcaaggtgaaactgtc 
               
               
                   
               
               
                 SEQ ID NO:211 
                 ctgaatcatgaaatgttctactctgc 
               
               
                   
               
               
                 SEQ ID NO:212 
                 tgtcaacttgactgggccatacg 
               
               
                   
               
               
                 SEQ ID NO:213 
                 ctcccgtatagttgggattatagg 
               
               
                   
               
               
                 SEQ ID NO:214 
                 gcttggagttccttgaaattcttgg 
               
               
                   
               
               
                 SEQ ID NO:215 
                 cctggtggctccagttttctacc 
               
               
                   
               
               
                 SEQ ID NO:216 
                 aactcctgacctcatgatccacc 
               
               
                   
               
               
                 SEQ ID NO:217 
                 gctgggattacaggcatgagcc 
               
               
                   
               
               
                 SEQ ID NO:218 
                 ttctcctttatccttggtgacattc 
               
               
                   
               
               
                 SEQ ID NO:219 
                 tcccaaagtgctgggattacagg 
               
               
                   
               
               
                 SEQ ID NO:220 
                 gtcataagtcagggaccatctgc 
               
               
                   
               
               
                 SEQ ID NO:221 
                 ctgtttcattgatttccagactggc 
               
               
                   
               
               
                 SEQ ID NO:222 
                 gcaatctcggctcactgcaagc 
               
               
                   
               
               
                 SEQ ID NO:223 
                 gaagaagtgactatatcagatctgg 
               
               
                   
               
               
                 SEQ ID NO:224 
                 ttcaccatgttggccaggctgg 
               
               
                   
               
               
                 SEQ ID NO:225 
                 catcactgaagatgacaactgagc 
               
               
                   
               
               
                 SEQ ID NO:226 
                 gtccagcctgggcgatagagc 
               
               
                   
               
               
                 SEQ ID NO:227 
                 gaggaaagtctttgaagaggaacc 
               
               
                   
               
               
                 SEQ ID NO:228 
                 ggtacactcaccagcagttttgc 
               
               
                   
               
               
                 SEQ ID NO:229 
                 gagcaactggtgtgaatacatatgg 
               
               
                   
               
               
                 SEQ ID NO:230 
                 caatacctggcaccacatacacc 
               
               
                   
               
               
                 SEQ ID NO:231 
                 gggactacaggcatgtgccacc 
               
               
                   
               
               
                 SEQ ID NO:232 
                 cggtggctcacgcgtgtaatcc 
               
               
                   
               
               
                 SEQ ID NO:233 
                 caactgttaaatctctcatggaaacc 
               
               
                   
               
               
                 SEQ ID NO:234 
                 gacaaaggattagaaatgcaccc 
               
               
                   
               
               
                 SEQ ID NO:235 
                 ggaaatgttctaaaactggattgtgg 
               
               
                   
               
               
                 SEQ ID NO:236 
                 aataataatagccaggtgtggtagc 
               
               
                   
               
               
                 SEQ ID NO:237 
                 ctggaacactcacacattgctgg 
               
               
                   
               
               
                 SEQ ID NO:235 
                 ctgggtgacagagcgagactcc 
               
               
                   
               
               
                 SEQ ID NO:239 
                 cccaaatcatccccgtgaaacatgc 
               
               
                   
               
               
                 SEQ ID NO:240 
                 gaccctgcaatcccaacactgg 
               
               
                   
               
               
                 SEQ ID NO:241 
                 ctctcaggccttcaaactacacc 
               
               
                   
               
               
                 SEQ ID NO:242 
                 caggaaagggctcgctcagtgg 
               
               
                   
               
               
                 SEQ ID NO:243 
                 atctgcaaaagcagcagagcagg 
               
               
                   
               
               
                 SEQ ID NO:244 
                 gtacccatgacagacaagttttagg 
               
               
                   
               
               
                 SEQ ID NO:245 
                 cttatcccctactgtctcctttgg 
               
               
                   
               
               
                 SEQ ID NO:246 
                 ggatggtctcgatctcctgacc 
               
               
                   
               
               
                 SEQ ID NO:247 
                 aggttagagaccttcctctaatgc 
               
               
                   
               
               
                 SEQ ID NO:248 
                 agctgggattacaggtgcctgc 
               
               
                   
               
               
                 SEQ ID NO:249 
                 gctgaggcaggttggggctgc 
               
               
                   
               
               
                 SEQ ID NO:250 
                 acatttaacgtctcctaacttctcc 
               
               
                   
               
               
                 SEQ ID NO:251 
                 gtgctgcgattacaggtgtgagc 
               
               
                   
               
               
                 SEQ ID NO:252 
                 tatgacagcagtattatactatcacc 
               
               
                   
               
               
                 SEQ ID NO:253 
                 ctggggaccaaatctgaactgcc 
               
               
                   
               
               
                 SEQ ID NO:254 
                 gtagctattgttatttccaaaagagg 
               
               
                   
               
               
                 SEQ ID NO:255 
                 gcttgggaccccaggacaagg 
               
               
                   
               
               
                 SEQ ID NO:256 
                 cctggccaacatggggaaatcc 
               
               
                   
               
               
                 SEQ ID NO:257 
                 aattgcttgaacctgggaggtgg 
               
               
                   
               
               
                 SEQ ID NO:258 
                 gcctaagacccaaaagctattagc 
               
               
                   
               
               
                 SEQ ID NO:259 
                 catattaaagggccatattcaaattgg 
               
               
                   
               
               
                 SEQ ID NO:260 
                 ggatgtaaccagtgtatatcacagg 
               
               
                   
               
               
                 SEQ ID NO:261 
                 ggaagtttagtccacatcttctagc 
               
               
                   
               
               
                 SEQ ID NO:262 
                 gcacccacaggacaaccacacg 
               
               
                   
               
               
                 SEQ ID NO:263 
                 gggacgcgcctgttaacaaagg 
               
               
                   
               
               
                 SEQ ID NO:264 
                 gggctgggggccacgctcc 
               
               
                   
               
               
                 SEQ ID NO:265 
                 cgcaaaagtgaagccctcctgg 
               
               
                   
               
               
                 SEQ ID NO:266 
                 gaaatcctacttgatctaaagtgagc 
               
               
                   
               
               
                 SEQ ID NO:267 
                 tttgagcaacttggaaaaaataagcg 
               
               
                   
               
               
                 SEQ ID NO:268 
                 ttcccaaaagacaaatagcacttcc 
               
               
                   
               
               
                 SEQ ID NO:269 
                 ccattttgaaaatcacagtgaattcc 
               
               
                   
               
               
                 SEQ ID NO:270 
                 gaaaagaaaaccctgaattcaaaagg 
               
               
                   
               
               
                 SEQ ID NO:271 
                 tgctgaaaagaagcatttaaaagtgg 
               
               
                   
               
               
                 SEQ ID NO:272 
                 ctcttaccagtttcagagctttcc 
               
               
                   
               
               
                 SEQ ID NO:273 
                 ttttcagccaaaaatcaaggacagg 
               
               
                   
               
               
                 SEQ ID NO:274 
                 cttgagcccaggagtttgagacc 
               
               
                   
               
               
                 SEQ ID NO:275 
                 cgcctgtagtaccctctactagg 
               
               
                   
               
               
                 SEQ ID NO:276 
                 ggtaaagaaagaaggatttgaaaacc 
               
               
                   
               
               
                 SEQ ID NO:277 
                 taagagtaatgaggttaaagtttatgc 
               
               
                   
               
               
                 SEQ ID NO:278 
                 catttttattgtcacaggccatttgc 
               
               
                   
               
               
                 SEQ ID NO:279 
                 gccacgccttctcttctgccacc 
               
               
                   
               
               
                 SEQ ID NO:280 
                 tgcctctcctgactgcactgtg 
               
               
                   
               
               
                 SEQ ID NO:281 
                 ccatgctctaccacgcccttgg 
               
               
                   
               
               
                 SEQ ID NO:282 
                 cattcaggctggagtgcggtgg 
               
               
                   
               
               
                 SEQ ID NO:283 
                 cttaaaaattgtctggctaagacattg 
               
               
                   
               
               
                 SEQ ID NO:284 
                 ttgctcttgttgcccgggttgg 
               
               
                   
               
               
                 SEQ ID NO:285 
                 gagcttagaggaaaagtattatttcc 
               
               
                   
               
               
                 SEQ ID NO:286 
                 tggtgctgtgccagacgctgg 
               
               
                   
               
               
                 SEQ ID NO:287 
                 cagatctttttggctattgtcttgg 
               
               
                   
               
               
                 SEQ ID NO:288 
                 gaaggaaagggcctcccactgc 
               
               
                   
               
               
                 SEQ ID NO:289 
                 catgaaaaagcatgctggggagg 
               
               
                   
               
               
                 SEQ ID NO:290 
                 caaacataaaaaagctttaatagaagcc 
               
               
                   
               
               
                 SEQ ID NO:291 
                 tcccaactatgaaaaaatagaagacg 
               
               
                   
               
               
                 SEQ ID NO:292 
                 cacaaattagccgggcatggtgg 
               
               
                   
               
               
                 SEQ ID NO:293 
                 cttcctttactgagtctttctaaagc 
               
               
                   
               
               
                 SEQ ID NO:294 
                 tgtcctttgaaatgtaggtatgtgg 
               
               
                   
               
               
                 SEQ ID NO:295 
                 ggatcttgcaatactgacatctcc 
               
               
                   
               
               
                 SEQ ID NO:296 
                 atttgaaaagaactgaaggatctacc 
               
               
                   
               
               
                 SEQ ID NO:297 
                 gtgagctgagatctcgtctctgc 
               
               
                   
               
               
                 SEQ ID NO:298 
                 tttgtctgaaacagattctaaaagttgg 
               
               
                   
               
               
                 SEQ ID NO:299 
                 gcaggtgcctgtagtcccagc 
               
               
                   
               
               
                 SEQ ID NO:300 
                 gtttgagcttctaaaattcatggattc 
               
               
                   
               
               
                 SEQ ID NO:301 
                 gtggtaggtcaaaccgcaattcc 
               
               
                   
               
               
                 SEQ ID NO:302 
                 accaaatcagacatatcagctttgg 
               
               
                   
               
               
                 SEQ ID NO:303 
                 cacagaacggatcctcaataaagg 
               
               
                   
               
               
                 SEQ ID NO:304 
                 gttaactcctcccttctctttatgg 
               
               
                   
               
               
                 SEQ ID NO:305 
                 gtgttcagagagcttgatttccagg 
               
               
                   
               
               
                 SEQ ID NO:306 
                 cccacttgatttttcccacatgg 
               
               
                   
               
               
                 SEQ ID NO:307 
                 atttatttagatgaagtgaatattttcc 
               
               
                   
               
               
                 SEQ ID NO:308 
                 atttagtttgtttaactgtgagtgc 
               
               
                   
               
               
                 SEQ ID NO:309 
                 gtacagaagtgcttgatgcatacc 
               
               
                   
               
               
                 SEQ ID NO:310 
                 aggcagataaaaattctccattagc 
               
               
                   
               
               
                 SEQ ID NO:311 
                 acaagcacgagccacagcacc 
               
               
                   
               
               
                 SEQ ID NO:312 
                 cgctcttgttgcccaggctgg 
               
               
                   
               
               
                 SEQ ID NO:313 
                 cccaaaacagactttctagataacc 
               
               
                   
               
               
                 SEQ ID NO:314 
                 ttcaaattgctttttttctactcacc 
               
               
                   
               
               
                 SEQ ID NO:315 
                 gatctgaaaaaagtgacaggttgg 
               
               
                   
               
               
                 SEQ ID NO:316 
                 cactgaaatttgaaaggaacatatgg 
               
               
                   
               
               
                 SEQ ID NO:317 
                 tctggtgcagtggcctctagg 
               
               
                   
               
               
                 SEQ ID NO:318 
                 accataagtggttttacctgatgg 
               
               
                   
               
               
                 SEQ ID NO:319 
                 cccaggcgcaggtgattctcc 
               
               
                   
               
               
                 SEQ ID NO:320 
                 ggtggctcacgcctgaaatcc 
               
               
                   
               
               
                 SEQ ID NO:321 
                 cacagtccacgtgccacaatcc 
               
               
                   
               
               
                 SEQ ID NO:322 
                 aatcatgttaacacatccctctcc 
               
               
                   
               
               
                 SEQ ID NO:323 
                 gaagagagtgttgaaaggttaagc 
               
               
                   
               
               
                 SEQ ID NO:324 
                 cgagaccatactggctaagatgg 
               
               
                   
               
               
                 SEQ ID NO:325 
                 attagccacacaataaatgttctgg 
               
               
                   
               
               
                 SEQ ID NO:326 
                 tttgaaaagcgttgcaatatgatgc 
               
               
                   
               
               
                 SEQ ID NO:327 
                 ggttgcagtgagccgagatcg 
               
               
                   
               
               
                 SEQ ID NO:328 
                 ggtgggaggactgcctgagc 
               
               
                   
               
               
                 SEQ ID NO:329 
                 aacagagagaaaaaacacaaattacc 
               
               
                   
               
               
                 SEQ ID NO:330 
                 gatatctagaattcccaaatacttgg 
               
               
                   
               
               
                 SEQ ID NO:331 
                 gtgatagaattaaaggaaaaaataaacg 
               
               
                   
               
               
                 SEQ ID NO:332 
                 attgttccttttctaaatattctacc 
               
               
                   
               
               
                 SEQ ID NO:333 
                 cagcactttgggaggctgagg 
               
               
                   
               
               
                 SEQ ID NO:334 
                 cacagaggtttcacagtgctgg 
               
               
                   
               
               
                 SEQ ID NO:335 
                 aacttctgcttctgtccataatgc 
               
               
                   
               
               
                 SEQ ID NO:336 
                 gcctgtaatcccagcactttgg 
               
               
                   
               
               
                 SEQ ID NO:337 
                 gccagtaaacatatgaaaaggtgc 
               
               
                   
               
               
                 SEQ ID NO:338 
                 aattatgtaaataaagagtgaaaagg 
               
               
                   
               
               
                 SEQ ID NO:339 
                 cccctacacagaaaaaacaattcc 
               
               
                   
               
               
                 SEQ ID NO:340 
                 tgagtgtcaaagaaaaatacaattgg 
               
               
                   
               
               
                 SEQ ID NO:341 
                 atacacagagaaaatgagtccacc 
               
               
                   
               
               
                 SEQ ID NO:342 
                 aacactccccttctctgtttagc 
               
               
                   
               
               
                 SEQ ID NO:343 
                 gatattctttgcaacctaggatgc 
               
               
                   
               
               
                 SEQ ID NO:344 
                 ctctaaaactaatcagcaatgtaacc 
               
               
                   
               
               
                 SEQ ID NO:345 
                 cacctgtaatcccagcactttgg 
               
               
                   
               
               
                 SEQ ID NO:346 
                 cgtaaaactgccacaaagcttgtagg 
               
               
                   
               
               
                 SEQ ID NO:347 
                 gtggcagaggtgcaagcaagc 
               
               
                   
               
               
                 SEQ ID NO:348 
                 acagaaatgacaaacgcatgtacc 
               
               
                   
               
               
                 SEQ ID NO:349 
                 acactctcttagctaggctttgg 
               
               
                   
               
               
                 SEQ ID NO:350 
                 gagcttggaatagggcagttcc 
               
               
                   
               
               
                 SEQ ID NO;351 
                 ctgggttctttaaacatgtccagg 
               
               
                   
               
               
                 SEQ ID NO:352 
                 tcaagaaaggacactgcagtggc 
               
               
                   
               
               
                 SEQ ID NO:353 
                 catgcacacaaactatctcattcc 
               
               
                   
               
               
                 SEQ ID NO:354 
                 tagccgggcatggtggcacg 
               
               
                   
               
               
                 SEQ ID NO:355 
                 atcatgctgattgaatttcaaatagc 
               
               
                   
               
               
                 SEQ ID NO:356 
                 ttggcatgcagggcagtgacc 
               
               
                   
               
               
                 SEQ ID NO:357 
                 ggtggtgagataataacacctgc 
               
               
                   
               
               
                 SEQ ID NO:358 
                 ttgctatataataatcatttgtgatcc 
               
               
                   
               
               
                 SEQ ID NO:359 
                 cggtaactgttactctgggatgg 
               
               
                   
               
               
                 SEQ ID NO:360 
                 aggctaggttcccttctcttcc 
               
               
                   
               
               
                 SEQ ID NO:361 
                 gtagtgcctagcacagagaaagc 
               
               
                   
               
               
                 SEQ ID NO:362 
                 ctagcctgggcaacaagagcg 
               
               
                   
               
               
                 SEQ ID NO:363 
                 tctctctcctctctgggatcag 
               
               
                   
               
               
                 SEQ ID NO:364 
                 gtttgaatatttgtatgcagcaagc 
               
               
                   
               
               
                 SEQ ID NO:365 
                 tagaacaaattctggcttataaaagc 
               
               
                   
               
               
                 SEQ ID NO:366 
                 ccactctacctttattccttgcc 
               
               
                   
               
               
                 SEQ ID NO:367 
                 agaccagaatatgcaagcagagg 
               
               
                   
               
               
                 SEQ ID NO:368 
                 ggacgttttgctggtgtctgcg 
               
               
                   
               
               
                 SEQ ID NO:369 
                 aaggaacaaactgttgtcacatgc 
               
               
                   
               
               
                 SEQ ID NO:370 
                 atgtagctgggactacaggtgc 
               
               
                   
               
               
                 SEQ ID NO:371 
                 ggctcatgcctgtaatcccagc 
               
               
                   
               
               
                 SEQ ID NO:372 
                 atgaggttttcacacaaaaagatgc 
               
               
                   
               
               
                 SEQ ID NO:373 
                 tgggcgacagagcaagactcc 
               
               
                   
               
               
                 SEQ ID NO:374 
                 aaatgtccctaaaagtgatcaacagc 
               
               
                   
               
               
                 SEQ ID NO:375 
                 cagactcagttttacctcatcagc 
               
               
                   
               
               
                 SEQ ID NO:376 
                 agtgatctttcctctttaacctcc 
               
               
                   
               
               
                 SEQ ID NO:377 
                 ccagctattcaggaggccaagg 
               
               
                   
               
               
                 SEQ ID NO:378 
                 cttaaacattatgacactgtcttgc 
               
               
                   
               
               
                 SEQ ID NO:379 
                 ccaggtctatgaggccgttcc 
               
               
                   
               
               
                 SEQ ID NO:380 
                 tccaaagcatccctacattatacc 
               
               
                   
               
               
                 SEQ ID NO:381 
                 acatacatacatgcagtgactagc 
               
               
                   
               
               
                 SEQ ID NO:382 
                 tacaggtgccagccaccatgc 
               
               
                   
               
               
                 SEQ ID NO:383 
                 gcctgtaatcccagcactctgg 
               
               
                   
               
               
                 SEQ ID NO:384 
                 gacagagtcccactcttgttgc 
               
               
                   
               
               
                 SEQ ID NO:385 
                 gtgccttccaaagcagtgtagg 
               
               
                   
               
               
                 SEQ ID NO:386 
                 tatcttactgggtatgtataatgcc 
               
               
                   
               
               
                 SEQ ID NO:387 
                 caaaggaaatacgtcctaccagg 
               
               
                   
               
               
                 SEQ ID NO:388 
                 ccttttctcacagacatgcttcc 
               
               
                   
               
               
                 SEQ ID NO:389 
                 taaacacagtgagcagaatccc 
               
               
                   
               
               
                 SEQ ID NO:390 
                 ataaagcaaacttctaaaagggtcc 
               
               
                   
               
               
                 SEQ ID NO:391 
                 accactacactccagcctggg 
               
               
                   
               
               
                 SEQ ID NO:392 
                 gatacctgggtcagagtaagtgc 
               
               
                   
               
               
                 SEQ ID NO:393 
                 tgtaatctcagctacttgggagg 
               
               
                   
               
               
                 SEQ ID NO:394 
                 gtgtcgtcttctcttcctctacg 
               
               
                   
               
               
                 SEQ ID NO:395 
                 ctggctagtatgaggttggtgc 
               
               
                   
               
               
                 SEQ ID NO:396 
                 ggactagccacatttcaaccagg 
               
               
                   
               
               
                 SEQ ID NO:397 
                 gcagtatactgagaatttagtttcc 
               
               
                   
               
               
                 SEQ ID NO:398 
                 gaggctgaggcaggagaatgg 
               
               
                   
               
               
                 SEQ ID NO:399 
                 cattgtttgatgaaggtcaacagc 
               
               
                   
               
               
                 SEQ ID NO:400 
                 cagacaagagtggctacggcag 
               
               
                   
               
               
                 SEQ ID NO:401 
                 acgcccagccagattattcagg 
               
               
                   
               
               
                 SEQ ID NO:402 
                 ggaaccagaaagaagtgcaaagg 
               
               
                   
               
               
                 SEQ ID NO:403 
                 tgagccatcttggaggcaggc 
               
               
                   
               
               
                 SEQ ID NO:404 
                 caggaccttcctacaaacctcc 
               
               
                   
               
               
                 SEQ ID NO:405 
                 aacacaacatatctgaccttacgc 
               
               
                   
               
               
                 SEQ ID NO:406 
                 gccttagaagtccagaggaaagc 
               
               
                   
               
               
                 SEQ ID NO:407 
                 tgacgtacccagtagaccttcc 
               
               
                   
               
               
                 SEQ ID NO:408 
                 ctctgcaagcctgggaaacagg 
               
               
                   
               
               
                 SEQ ID NO:409 
                 gccttgtccccaagtcctaagg 
               
               
                   
               
               
                 SEQ ID NO:410 
                 gcaaagggactcctggaattcc 
               
               
                   
               
               
                 SEQ ID NO:411 
                 gctcctgcctgtaatcccagc 
               
               
                   
               
               
                 SEQ ID NO:412 
                 gaaggaaacagaaaaagcagaggc 
               
               
                   
               
               
                 SEQ ID NO:413 
                 cttactaccgttcttcttcactgg 
               
               
                   
               
               
                 SEQ ID NO:414 
                 actattctgtttctttaggtttactgc 
               
               
                   
               
               
                 SEQ ID NO:415 
                 cggtggctcacacctgtaatcc 
               
               
                   
               
               
                 SEQ ID NO:416 
                 agccagagttctgtgctctagg 
               
               
                   
               
               
                 SEQ ID NO:417 
                 taatttgcatttcgtgccgctcc 
               
               
                   
               
               
                 SEQ ID NO:418 
                 cacttttaatacagatcccaatagg 
               
               
                   
               
               
                 SEQ ID NO:419 
                 atgtattttttcttttcctgtcaagc 
               
               
                   
               
               
                 SEQ ID NO:420 
                 aaatgttaacattattctccctaagg 
               
               
                   
               
               
                 SEQ ID NO:421 
                 catatgcccagatcccgtctcc 
               
               
                   
               
               
                 SEQ ID NO:422 
                 acaggtgtgagccgctgcacc 
               
               
                   
               
               
                 SEQ ID NO:423 
                 gccaagacgtttacagttttggc 
               
               
                   
               
               
                 SEQ ID NO:424 
                 aggaaacttctgaggatgatggg 
               
               
                   
               
               
                 SEQ ID NO:425 
                 gctttatagggcagtctgaattcc 
               
               
                   
               
               
                 SEQ ID NO:426 
                 ttagaataaaagttatctcgggagg 
               
               
                   
               
               
                 SEQ ID NO:427 
                 taatttcttcagctttatccctcag 
               
               
                   
               
               
                 SEQ ID NO:428 
                 cacatgactaattctctattcattcc 
               
               
                   
               
               
                 SEQ ID NO:429 
                 aaagacctcaagaaaagagtcacc 
               
               
                   
               
               
                 SEQ ID NO:430 
                 gacccataaagattatatgcccag 
               
               
                   
               
               
                 SEQ ID NO:431 
                 aaagtactaatgcagtgtgtcagc 
               
               
                   
               
               
                 SEQ ID NO:432 
                 gaggttcctcgattcccctgc 
               
               
                   
               
               
                 SEQ ID NO:433 
                 ggagagcagaggaattcacagg 
               
               
                   
               
               
                 SEQ ID NO:434 
                 agtaattagaaactgattctaagacg 
               
               
                   
               
               
                 SEQ ID NO:435 
                 cataccattgccaatccagttcc 
               
               
                   
               
               
                 SEQ ID NO:436 
                 attacgggtgcctgccactgc 
               
               
                   
               
               
                 SEQ ID NO:437 
                 cagccaggcagaggagagagg 
               
               
                   
               
               
                 SEQ ID NO:438 
                 ttttcattccaagtttctgtttggg 
               
               
                   
               
               
                 SEQ ID NO:439 
                 tttcaaataggaatttggataatccc 
               
               
                   
               
               
                 SEQ ID NO:440 
                 taagccgagatcacaccactgc 
               
               
                   
               
               
                 SEQ ID NO:441 
                 ccttcagcgcattatatcttggc 
               
               
                   
               
               
                 SEQ ID NO:442 
                 ccatctaatccatcttaaattcacc 
               
               
                   
               
               
                 SEQ ID NO:443 
                 gagtggagactgcgccactgc 
               
               
                   
               
               
                 SEQ ID NO:444 
                 aatcatgtgccaattaaaccatggc 
               
               
                   
               
               
                 SEQ ID NO:445 
                 cccagggaccagaccagacc 
               
               
                   
               
               
                 SEQ ID NO:446 
                 ctcactcaccagtgaaaatcagc 
               
               
                   
               
               
                 SEQ ID N0:447 
                 ggttgctctcgaactcctgacc 
               
               
                   
               
               
                 SEQ ID NO:448 
                 gttcccccagctcctttctgc 
               
               
                   
               
               
                 SEQ ID NO:449 
                 agaaagatgtagaagggtccagc 
               
               
                   
               
               
                 SEQ ID NO:450 
                 gggaaaaggtgtattatgcaagcg 
               
               
                   
               
               
                 SEQ ID NO:451 
                 ctctctcagacctaatgcaaaagc 
               
               
                   
               
               
                 SEQ ID NO:452 
                 aactatacatacagtatttgtattagc 
               
               
                   
               
               
                 SEQ ID NO:453 
                 aaattaatgcaatccatgatccagg 
               
               
                   
               
               
                 SEQ ID NO:454 
                 ctttctccactciaagagaaccc 
               
               
                   
               
               
                 SEQ ID NO:455 
                 ttttggtgtgttcatattggctgc 
               
               
                   
               
               
                 SEQ ID NO:456 
                 gcttccacaaatgacagacaaagg 
               
               
                   
               
               
                 SEQ ID NO:457 
                 ggctcatgcttgtaatcccagc 
               
               
                   
               
               
                 SEQ ID NO:458 
                 catatgaattgttgttcctttgtagg 
               
               
                   
               
               
                 SEQ ID NO:459 
                 cactggtacaagtccaagagtcc 
               
               
                   
               
               
                 SEQ ID NO:460 
                 gaccctgtgtctacttcctggg 
               
               
                   
               
               
                 SEQ ID NO:461 
                 tatttgaactatctcttgaaatgtcc 
               
               
                   
               
               
                 SEQ ID NO:462 
                 ctgattaaaaagtattacccttggc 
               
               
                   
               
               
                 SEQ ID NO:463 
                 tttgaaactgcactcaataacttgg 
               
               
                   
               
               
                 SEQ ID NO:464 
                 agtaatgtgtcatgatccaatggc 
               
               
                   
               
               
                 SEQ ID NO:465 
                 gaaagcatttcccaatgtctcacc 
               
               
                   
               
               
                 SEQ ID NO:466 
                 caatggacaaaaggcccaactgc 
               
               
                   
               
               
                 SEQ ID NO:467 
                 tccagctctggcttttttgttaag 
               
               
                   
               
               
                 SEQ ID NO:468 
                 acggagtctcactccgtgacc 
               
               
                   
               
               
                 SEQ ID NO:469 
                 ctatgtcatagtcaagagactttgc 
               
               
                   
               
               
                 SEQ ID NO:470 
                 gttcaagcgattctcctgtctcg 
               
               
                   
               
               
                 SEQ ID NO:471 
                 ccacctaatacttaaatacggaagc 
               
               
                   
               
               
                 SEQ ID NO:472 
                 atattcaacaaacttaatagtgaagtg 
               
               
                   
               
               
                 SEQ ID NO:473 
                 ttacaggcgtgagtcaccatgc 
               
               
                   
               
               
                 SEQ ID NO:474 
                 aacacctccaagaggccaaacg 
               
               
                   
               
               
                 SEQ ID NO:475 
                 tactattggcaaatttcaattatatgg 
               
               
                   
               
               
                 SEQ ID NO:476 
                 agcccacatcctaaaattcaataag 
               
               
                   
               
               
                 SEQ ID NO:477 
                 gaaagtggataagtgtttgtctgg 
               
               
                   
               
               
                 SEQ ID NO:478 
                 ggccaggcattcaagaccagc 
               
               
                   
               
               
                 SEQ ID NO:479 
                 agccaacaacaaaaagacacaacc 
               
               
                   
               
               
                 SEQ ID NO:480 
                 ttgagcccaggagttcaagacc 
               
               
                   
               
               
                 SEQ ID NO:481 
                 cagactaaagatctcagagagaaac 
               
               
                   
               
               
                 SEQ ID NO:482 
                 cgcttgtaatcccagcacttgg 
               
               
                   
               
               
                 SEQ ID NO:483 
                 aaaagtgaaatcagaatttgtttcc 
               
               
                   
               
               
                 SEQ ID NO:484 
                 caggcgtgagcaactgtgtcc 
               
               
                   
               
               
                 SEQ ID NO:485 
                 ggtccagtaggatctcgtttgc 
               
               
                   
               
               
                 SEQ ID NO:486 
                 actttgaaaatgttgttatagctggg 
               
               
                   
               
               
                 SEQ ID NO:487 
                 ttccctgcatctaagtcttctcc 
               
               
                   
               
               
                 SEQ ID NO:488 
                 agatatctaccattgaagagtttgc 
               
               
                   
               
               
                 SEQ ID NO:489 
                 agtcttcacttcactttgttgtcc 
               
               
                   
               
               
                 SEQ ID NO:490 
                 ccatgcaggtatgaaatataaaagc 
               
               
                   
               
               
                 SEQ ID NO:491 
                 tgggtgacagagtgagactcc 
               
               
                   
               
               
                 SEQ ID NO:492 
                 acagcaataccgggttaacatgc 
               
               
                   
               
               
                 SEQ ID NO:493 
                 tttatgtaaaagatgaatgcgaggc 
               
               
                   
               
               
                 SEQ ID NO:494 
                 ctactctgctactgggaacagg 
               
               
                   
               
               
                 SEQ ID NO:495 
                 caaacgttagtctggcaaaatgcg 
               
               
                   
               
               
                 SEQ ID NO:496 
                 tgcacgctaccacacccagc 
               
               
                   
               
               
                 SEQ ID NO:497 
                 aattcttggatctgtgtgtttactgc 
               
               
                   
               
               
                 SEQ ID NO:498 
                 taccagttatcattctctttctgc 
               
               
                   
               
               
                 SEQ ID NO:499 
                 atccacccacctcggcctcc 
               
               
                   
               
               
                 SEQ ID NO:500 
                 cactctgcctggcccttaatgg 
               
               
                   
               
               
                 SEQ ID NO:501 
                 atagtttgtttaatatgccactaagg 
               
               
                   
               
               
                 SEQ ID NO:502 
                 gcgtgagccaccgcacctgg 
               
               
                   
               
               
                 SEQ ID NO:503 
                 ctccatcacacaaattttatgtggc 
               
               
                   
               
               
                 SEQ ID NO:504 
                 agacggagtctcgttctgtcgc 
               
               
                   
               
               
                 SEQ ID NO:505 
                 tcccaggttcaagccattctcc 
               
               
                   
               
               
                 SEQ ID NO:506 
                 tattttgagagtctcactctgtcg 
               
               
                   
               
               
                 SEQ ID NO:507 
                 gtctcgaactcctgacctcagg 
               
               
                   
               
               
                 SEQ ID NO:508 
                 aaggaggtgaagagtgaactacg 
               
               
                   
               
               
                 SEQ ID NO:509 
                 gtctcaggttttggacttacttgg 
               
               
                   
               
               
                 SEQ ID NO:510 
                 tttacagatcttaaatgcattaggac 
               
               
                   
               
               
                 SEQ ID NO:511 
                 gtacactgaacaaaggagacagg 
               
               
                   
               
               
                 SEQ ID NO:512 
                 ctggtagtaatgcaaaatagcacc 
               
               
                   
               
               
                 SEQ ID NO:513 
                 catttaatgtgaaatgaattataagcc 
               
               
                   
               
               
                 SEQ ID NO:514 
                 gagacagggtttcactatgttgg 
               
               
                   
               
               
                 SEQ ID NO:515 
                 ccagcactttggaaggctgagg 
               
               
                   
               
               
                 SEQ ID NO:516 
                 gaaaccaagtatcatggtaaattgc 
               
               
                   
               
               
                 SEQ ID NO:517 
                 cagtgagggctgctcagttcc 
               
               
                   
               
               
                 SEQ ID NO:518 
                 gccaggtgcggtggctcacg 
               
               
                   
               
               
                 SEQ ID NO:519 
                 catgcctgtaatcccagctacc 
               
               
                   
               
               
                 SEQ ID NO:520 
                 atgtaaatggtacagtcactttagg 
               
               
                   
               
               
                 SEQ ID NO:521 
                 cccacaatacagagaactcttacc 
               
               
                   
               
               
                 SEQ ID NO:522 
                 tgaaacatgcagcccagtgtcc 
               
               
                   
               
               
                 SEQ ID NO:523 
                 tgttttttctcctgccttcaatcc 
               
               
                   
               
               
                 SEQ ID NO:524 
                 gctttcctgggtctccatctgg 
               
               
                   
               
               
                 SEQ ID NO:525 
                 gcagccgcttgaaaacaaaacagc 
               
               
                   
               
               
                 SEQ ID NO:526 
                 gatcacgttacatttgggggtgg 
               
               
                   
               
               
                 SEQ ID NO:527 
                 taggctgaaaaactaaaatttgttgc 
               
               
                   
               
               
                 SEQ ID NO:528 
                 ctcctttgggctcctttagtcc 
               
               
                   
               
               
                 SEQ ID NO:529 
                 gcctcggcctcccaaagtgc 
               
               
                   
               
               
                 SEQ ID NO:530 
                 aatgcctagagagatttggcagg 
               
               
                   
               
               
                 SEQ ID NO:531 
                 gagatggggtttcactatgttgg 
               
               
                   
               
               
                 SEQ ID NO:532 
                 tgtgatcttgccactgcactcc 
               
               
                   
               
               
                 SEQ ID NO:533 
                 acttctcctccatttgtttcttcg 
               
               
                   
               
               
                 SEQ ID NO:534 
                 cgtgcccgggctcagttctac 
               
               
                   
               
               
                 SEQ ID NO:535 
                 ccaaaacaataaaatcacaatttggg 
               
               
                   
               
               
                 SEQ ID NO:536 
                 ctgaactgccttagagtaaatccg 
               
               
                   
               
               
                 SEQ ID NO:537 
                 atttctgtatcaggtctgtgttcc 
               
               
                   
               
               
                 SEQ ID NO:538 
                 ggctgaccccttcactgtttcc 
               
               
                   
               
               
                 SEQ ID NO:539 
                 caaaaattagccaggcatggtgg 
               
               
                   
               
               
                 SEQ ID NO:540 
                 gcagtgagcagtgatcgcacc 
               
               
                   
               
               
                 SEQ ID NO:541 
                 aaagactgtgaactaacttgtttgc 
               
               
                   
               
               
                 SEQ ID NO:542 
                 tgccaagaattacacattattaggc 
               
               
                   
               
               
                 SEQ ID NO:543 
                 ggccaggatgtcattaactttcc 
               
               
                   
               
               
                 SEQ ID NO:544 
                 gtaagagctgacgtgtattgtgc 
               
               
                   
               
               
                 SEQ ID NO:545 
                 cccggtgaggccgcacatcc 
               
               
                   
               
               
                 SEQ ID NO:546 
                 cctgcgccttaaccccctcc 
               
               
                   
               
               
                 SEQ ID NO:547 
                 cggcgcctaggggccatcg 
               
               
                   
               
               
                 SEQ ID NO:548 
                 acttaaggaaacgaacatgacacc 
               
               
                   
               
               
                 SEQ ID NO:549 
                 gagaccgagtcttgctgtgtcg 
               
               
                   
               
               
                 SEQ ID NO:550 
                 gtattaattgaagatgatttggaatgc 
               
               
                   
               
               
                 SEQ ID NO:551 
                 tctttaaaagactatcgctgaggc 
               
               
                   
               
               
                 SEQ ID NO:552 
                 aaaagagacatcagtagagcatcc 
               
               
                   
               
               
                 SEQ ID NO:553 
                 gttcatgttttctttgacgtctcc 
               
               
                   
               
               
                 SEQ ID NO:554 
                 tttcgaaagttcaggctgagtgc 
               
               
                   
               
               
                 SEQ ID NO:555 
                 gaccctcaaaacaatcctctaagg 
               
               
                   
               
               
                 SEQ ID NO:556 
                 caaaacacacttagaaacaaactgc 
               
               
                   
               
               
                 SEQ ID NO:557 
                 gcctgggcgacatagtgagacc 
               
               
                   
               
               
                 SEQ ID NO:558 
                 ggcaggagaatggcgtgaacc 
               
               
                   
               
               
                 SEQ ID NO:559 
                 tttgctcgttgcccaggctgg 
               
               
                   
               
               
                 SEQ ID NO:560 
                 gcaacttaatgtgatagaataatagc 
               
               
                   
               
               
                 SEQ ID NO:561 
                 cctccccttctgctgccagc 
               
               
                   
               
               
                 SEQ ID NO:562 
                 ccacaacaatgtaaactcctctgg 
               
               
                   
               
               
                 SEQ ID NO:563 
                 tactctccctagagttcgttccc 
               
               
                   
               
               
                 SEQ ID NO:564 
                 gggtccccctttggccattcc 
               
               
                   
               
               
                 SEQ ID NO:565 
                 gatcttggctcacttcaacctcc 
               
               
                   
               
               
                 SEQ ID NO:566 
                 aggggaaatatttaaaccttgg 
               
               
                   
               
               
                 SEQ ID NO:567 
                 aatgcaatggtgcatttacagagg 
               
               
                   
               
               
                 SEQ ID NO:568 
                 tcattttatctatttctacatggtcc 
               
               
                   
               
               
                 SEQ ID NO:569 
                 ggaagggaaatgcccatgaacc 
               
               
                   
               
               
                 SEQ ID NO:570 
                 agtgaacattttctgcagcctcc 
               
               
                   
               
               
                 SEQ ID NO:571 
                 caacaggacgtcaggcgatcc 
               
               
                   
               
               
                 SEQ ID NO:572 
                 ccttcaggctgtcctgaaaagg 
               
               
                   
               
               
                 SEQ ID NO:573 
                 agtctcactccatcgcccagg 
               
               
                   
               
               
                 SEQ ID NO:574 
                 actgtgaacagtagttaactcagg 
               
               
                   
               
               
                 SEQ ID NO:575 
                 gcatgcctgtaatccaagctgc 
               
               
                   
               
               
                 SEQ ID NO:576 
                 gaaacaattctcttttcacacttgc 
               
               
                   
               
               
                 SEQ ID NO:577 
                 ggctcatgcctgttatcccagc 
               
               
                   
               
               
                 SEQ ID NO:578 
                 agaagaagcttagtcatatgtttgg 
               
               
                   
               
               
                 SEQ ID NO:579 
                 cagatgcttgagccaaacaaatgg 
               
               
                   
               
               
                 SEQ ID NO:580 
                 ctggcagacagagtgagactcc 
               
               
                   
               
               
                 SEQ ID NO:581 
                 aatgtgtgaatattattcattacaggg 
               
               
                   
               
               
                 SEQ ID NO:582 
                 gcaggagaattgcttgaacctgg 
               
               
                   
               
               
                 SEQ ID NO:583 
                 ctttagtcaaattaaaacagtctatcc 
               
               
                   
               
               
                 SEQ ID NO:584 
                 gatttctatctcctgcaaccacc 
               
               
                   
               
               
                 SEQ ID NO:585 
                 ttcttgtgtaactactaaaaatctcc 
               
               
                   
               
               
                 SEQ ID NO:586 
                 aaagggtcttcataaggctaatgg 
               
               
                   
               
               
                 SEQ ID NO:587 
                 ctcttaaggattatttatatgaagacc 
               
               
                   
               
               
                 SEQ ID NO:588 
                 caggaggagccccagagc 
               
               
                   
               
               
                 SEQ ID NO:589 
                 tcctggggatggttggatgc 
               
               
                   
               
               
                 SEQ ID NO:590 
                 tgaccccacagagtttacacagc 
               
               
                   
               
               
                 SEQ ID NO:591 
                 agtcagggcaggctctgcc 
               
               
                   
               
               
                 SEQ ID NO:592 
                 tattttggccccatccagaaagc 
               
               
                   
               
               
                 SEQ ID NO:593 
                 cacccagagtacagctttgttcc 
               
               
                   
               
               
                 SEQ ID NO:594 
                 gaggagccccagagcctgc 
               
               
                   
               
               
                 SEQ ID NO:595 
                 tggggatggttggatgcttacc 
               
               
                   
               
               
                 SEQ ID NO:596 
                 cccacagagtttacacagcttgc 
               
               
                   
               
               
                 SEQ ID NO:597 
                 caggctctgcccactcacc 
               
               
                   
               
               
                 SEQ ID NO:598 
                 ccatccagaaagcccaaagcc 
               
               
                   
               
               
                 SEQ ID NO:599 
                 ccagagtacagctttgttcctcattc 
               
               
                   
               
               
                 SEQ ID NO:600 
                 gcagtacaaacaacgcacagcg 
               
               
                   
               
               
                 SEQ ID NO:601 
                 ctgccaccctccacagtccc 
               
               
                   
               
               
                 SEQ ID NO:602 
                 gccaagaccatgcatgcg 
               
               
                   
               
               
                 SEQ ID NO:603 
                 cccagggacaaagagactccc 
               
               
                   
               
               
                 SEQ ID NO:604 
                 caggaagcagacagtcttctagttcc 
               
               
                   
               
               
                 SEQ ID NO:605 
                 tgcctgtaatcccaacactttgg 
               
               
                   
               
               
                 SEQ ID NO:606 
                 tccctctggccaggatggg 
               
               
                   
               
               
                 SEQ ID NO:607 
                 atggggaatgggagtaggaagc 
               
               
                   
               
               
                 SEQ ID NO:608 
                 cagatcagttctcccctccagc 
               
               
                   
               
               
                 SEQ ID NO:609 
                 acaaaaaagaaacatgctcagagagg 
               
               
                   
               
               
                 SEQ ID NO:610 
                 tggtggcatgcatctgtagtcc 
               
               
                   
               
               
                 SEQ ID NO:611 
                 aggtgctctatagatgttagcatccc 
               
               
                   
               
               
                 SEQ ID NO:612 
                 ccaggacaggatggagatctgg 
               
               
                   
               
               
                 SEQ ID NO:613 
                 agggaacctgtgcattatccttgc 
               
               
                   
               
               
                 SEQ ID NO:614 
                 cagaagtcttgctttaaggaggagg 
               
               
                   
               
               
                 SEQ ID NO:615 
                 gggtacgtgaaactcaccaagg 
               
               
                   
               
               
                 SEQ ID NO:616 
                 cagagtgtggcaagcaaggg 
               
               
                   
               
               
                 SEQ ID NO:617 
                 aacattttaaaggtacaaataacgtggg 
               
               
                   
               
               
                 SEQ ID NO:618 
                 tagggagcaacagccattaagc 
               
               
                   
               
               
                 SEQ ID NO:619 
                 ggtgcactgtccagctctgg 
               
               
                   
               
               
                 SEQ ID NO:620 
                 actctcgctgaactcgcctgg 
               
               
                   
               
               
                 SEQ ID NO:621 
                 ctcggtctctggtggtacgc 
               
               
                   
               
               
                 SEQ ID NO:622 
                 gcaagaggtccgagctggg 
               
               
                   
               
               
                 SEQ ID NO:623 
                 ggaagaagtgaaacaagagatgaagg 
               
               
                   
               
               
                 SEQ ID NO:624 
                 cccagagaacaaaccggattagg 
               
               
                   
               
               
                 SEQ ID NO:625 
                 cccttcaaccttctccaatctgc 
               
               
                   
               
               
                 SEQ ID NO:626 
                 cccatgtccagtggtttaggg 
               
               
                   
               
               
                 SEQ ID NO:627 
                 gagattggtgggagacagatgg 
               
               
                   
               
               
                 SEQ ID NO:628 
                 cttctcagctcaaagttccagcg 
               
               
                   
               
               
                 SEQ ID NO:629 
                 gaatgggagagatgaccagagg 
               
               
                   
               
               
                 SEQ ID NO:630 
                 aagggcaagggggtatgtgg 
               
               
                   
               
               
                 SEQ ID NO:631 
                 ggaaggaagcatgggaacacc 
               
               
                   
               
               
                 SEQ ID NO:632 
                 ccatcaatgctctgtctgtctgg 
               
               
                   
               
               
                 SEQ ID NO:633 
                 gtgccgtgactgtgcttgg 
               
               
                   
               
               
                 SEQ ID NO:634 
                 acatcccattgacctcatcaagc