Patent Abstract:
The invention has disclosed a method for diagnosis of dentinogenesis imperfecta type II (DGI-II) and/or dentinogenesis imperfecta type II with deafness (DGI-II with deafness). Said method comprises the steps of detecting the DSPP gene, transcript and/or protein in said subject and comparing it with the normal DSPP gene, transcript and/or protein to determine whether there is any variation, wherein said variation indicates that the possibility of suffering DGI-II and/or DGI-II with deafness in said subject is higher than the normal population. The present invention also discloses the method and pharmaceutical composition for treating DGI-II and/or DGI-II with deafness.

Full Description:
FIELD OF INVENTION  
         [0001]    This invention relates to both biological engineering and medical fields. In particular, it relates to a method of diagnosing and treating dentinogenesis imperfecta type II using human dentin sialophosphoprotein or DSPP gene and the coded product, and a pharmaceutical composition containing DSPP gene and/or protein.  
         TECHNICAL BACKGROUND  
         [0002]    The odontoblasts produce the dentin, which consists in mature tooth or the tooth during tooth development phase. During dentinogenesis, the odontoblasts form dentinal tubules. Dentin cell processes in these tubules make dentin a living tissue. During the primary stage of dentinogenesis, the odontoblasts synthesize, secrete and reabsorb the dentin matrix components. Protein synthesis occurs within cells. Exocytosis and endocytosis occurs mainly in cell processes. The first material formed is unmineralized mantle dentin matrix, mainly including collagen secreted by cells and non-collagenous components. The fasciculata collagen fibers congregate to a ball structure. Due to the continual increase of new fibrils, collagen becomes closer and closer. As a result, these prophase collagen fibers change into collagen fibers. Thus predentin characterized by collagen matrix is formed. Later, the mineralization crystals gradually deposited to become dentin at some distance away from cells.  
           [0003]    The mature dentin contains more inorganic minerals than the bone. 65 wt. % of dentin are minerals, mostly hydroxyapatite crystals. Organic materials are 20%, mainly collagenous proteins and non-collagenous proteins. These collagens offer braces to the deposition of hydroxyapatite plate like crystalline.  
           [0004]    Type I collagen is predominant (about 97%) in dentin collagens, 10%-15% of which is type I collagen trimer. Different from other connective tissue, type III collagen is lacking in dentin. Moreover, there are types V and VI collagens in dentin, but the contents are small. Although the contents of non-collagenous proteins in dentin are small, there are various kinds. According to the source of proteins, the dentin noncollagenous proteins can be divided to four kinds: dentin specific protein, mineralized tissue specific protein, aspecific protein, and blood serum source protein (or dentin affinity protein). Dentin specific protein is the only one which is synthesized and secreted by odontoblasts and exists only in dentin. Mineralized tissue specific proteins means those that are found and exist not only in dentin but also in cementum and bone. They are synthesized and secreted by osteoblasts, odontoblasts and cementoblasts. The non-specific proteins exist both in dentin and other tissues, including parenchyma, and synthesized and secreted by odontoblasts and other kinds of cells. Blood serum source proteins are those which are synthesized by other cells in the body, mainly by liver cells, and secreted to serum. These proteins have a high affinity to dentin, though they are not synthesized by dentin. They can enter dentin by blood circulation, so they are also called dentin affinity proteins. Proteoglycans or PGS are other primary non-collagenous proteins in dentin. They are large covalent molecules formed by many anylose side chains and one core protein. These side chains are composed of repeating disaccharide chain units, each of which consists of one glycuronic acid and one Nacetamidoacetose. One function of PGS in dentinogenesis is to affect or even control the systematism of collagen skeleton in predentin. The dentin proteoglycans fixed on the solid bracket can induce the formation of hydroxyapatite in vivo and in physiologic pH and ionic condition in vitro. On the contrary, the liquid proteoglycans restrain the form of mineral components in vitro. The combination of PGS and Ca 2+  is the precondition of inducing the formation of hydroxyapatite.  
           [0005]    Dentinogenesis imperfecta or DGI is an autosomal dominant dental genetic disease that has a prevalence of {fraction (1/8000)}. There are three types according to clinical taxonomy (1)  (The number in brackets shows the relative literature.). Dentinogenesis imperfecta Type I is also named DGI-I. Except for dentinogenesis imperfecta, patients usually have osteogenesis imperfecta The pathogeny is broad mutations in collagen type I gene (2) . Type II or DGI-II is also called hereditary opalescent dentin. DGI-II has a relationship with the improper mineralization of dentin and its penetrance is nearly 100% (3) . Type III or DGI-III is also called dentinogenesis imperfecta Brandywine type or isolate hereditary opalescent dentin. It is a special hereditary opalescent dentin, only found in three isolates in Washington, D.C., the State of Maryland, USA. Witkop first reported this illness in 1956 (4)  and there is no related report in China till now. DGI-III has an obviously genetic heterogeneity. Its pathogeny is related to malamineralization. Because the gene causing DGI-I has been found and DGI-III is only found in the isolates in the State of Maryland, USA, DGI-II becomes the focus of tooth endodontics.  
           [0006]    The clinical symptoms and pathology changes of DGI-II are as follows. The malajustment and turbulence of mineralization result in embryonic layer dysplasia in dentin. Both the primary dentition and permanent dentition are affected, with a more serious damage in primary dentition. A predominant feature is a blue-gray or amber brown discoloration of the teeth. The improper mineralized dentin is soft and the crown is prone to be worn. Moreover, compensatory hyperplasia of matrix increases in improperly mineralized dentin, leading to small or obliterated pulp chambers. Radiographs reveal that the affected teeth have bulbous crowns, narrow roots and small or obliterated pulp chambers and root canals. The pathology shows that the enamel surface is normal, but hypoplasia and hypocalcification can be found in about ⅓ of the patients. The enamel dentin junction changes greatly. Some teeth have a non-obvious sector structure in the enamel dentin junction. However, others are especially obvious. Dentin is lamellar with nearly normal outerdentin and dentinal tubules having subdivisional branches. In other parts, the dentin is obviously abnormal. Some short tubules or tubules with abnormal form distribute in dentin matrix disorderly. The predentin zone is very wide. Along the plywood, the remaining embedded cell can be seen, similar to embedded odontoblast and bloods. Observation under electron microscope indicates that the form and size of hypoplastic dentin micro-crystal are unchanged, but the quantity is small. Uncalcified or partly calcified transverse collagen fasciculi and volumes of crystal space can be seen discontinuously.  
           [0007]    For the mapping of DGI-II gene, in 1969, Bixler et al.( 5 ) tried to use some protein polymorphic markers, such as ABO, Rh, MNSs, Ke11, Fy, JK, HP, ACP1, PGM1 and PTC, to perform a linkage analysis in DGI-II families, but they failed to get the linkage evidence. In 1977, Mikkelsen et al. (6)  mapped a group of specific components (GC) in Vitamin D conjugated protein to 4q11-q13. In the next year, Kühnl identified that GC included six phenotypes: GC2/2, 2/1+, 2/1−, 1+/1−, 1+/1+, and 1−/1−. Later, Ball. S. P. et al. (7)  analyzed the linkage in a DGI-II big family named Family MRC4000 with the polymorphic markers of GC and found that DGI-II had a close linkage with GC (Lod=+7.9, θ=0.13). In 1992, Crall et al. (8)  mapped DGI-II to interval defined by two protein polymorphic markers: GC and interferon-inducible cytokine INP-10. The relative chromosome location was 4q12-21. The results above only offered a gross orientation of disease gene of DGI-II. Under that condition, it was almost impossible to clone the disease gene in this region.  
           [0008]    In 1995, Crosby A.H et al (9)  analyzed the linkage in two big DGI-II families with 9 short tandem repeat polymorphic markers (STRP) and mapped the disease gene to the 4q21-23 region defined by two STRPs of D4S2691 and D4S2692. Multipoints linkage analysis suggested that the disease gene might be in the region within about 3.2 cM around SPP1. Recently, Aplin H. M et al. (10)  genotyped two big families used by Crosby A. H with 5 hyperdense STRPs. The linkage analysis showed that the disease gene of DGIII located between two STRPs of GATA62A11 and D4S1563 with a genetic distance of 2 cM. Moreover, this research group established the YACs Contigs in this region. They also identified that DMP1, IBSP, SPP1 and DSPP are all in this candidate region by PCR technology.  
           [0009]    However, the mechanism of dentinogenesis imperfecta type II is still unclear so far. Also the direct relationship between dentinogenesis imperfecta type II and some special kind of protein is not reported.  
           [0010]    In addition, there is still no effective method to diagnose DGI-II early and/or antenatally and to cure DGI-II by non-operative treatment in the art.  
           [0011]    Therefore, there is an urgent need to develop new and efficient methods to diagnose and cure DGI-II, the relative pharmaceuticals, and diagnostic technology and reagents.  
         SUMMARY OF INVENTION  
         [0012]    One purpose of the invention is to provide a new diagnostic method and detection kit, especially for antenatal and/or early diagnosis of dentinogenesis imperfecta type II (DGI-II) and dentinogenesis imperfecta type II with deafness (DGI-II with deafness).  
           [0013]    Another purpose is to provide a new method to treat DGI-II and DGI-II with deafness.  
           [0014]    Still another purpose is to provide a pharmaceutical composition to treat DGI-II and DGI-II with deafness.  
           [0015]    In the first aspect, the invention provides a method for determining the susceptibility of DGI-II and/or DGI-II with deafness in a subject comprising the steps of:  
           [0016]    detecting the DSPP gene, transcript and/or protein in said subject and comparing it with the normal DSPP gene, transcript and/or protein to determine whether there is any difference,  
           [0017]    wherein said difference indicates that the possibility of suffering DGI-II and/or DGI-II with deafness in said subject is higher than the normal population.  
           [0018]    In a preferred embodiment, the DSPP gene or transcript is detected, and compared with the normal DSPP nucleotide sequence to determine the difference. More preferably, said difference is selected from the group consisting of: in position 1 of Exon 3, G1→T1; in position 1 of Intron 3, G1→A1.  
           [0019]    In the second aspect, the invention provides a method for treating DGI-II and/or DGI-II with deafness comprising the step of administrating a safe and effective amount of normal DSPP and/or DSP protein to the patient in need of said treatment. Preferably, the DSPP and/or DSP protein are administrated topically to periodontal tissues.  
           [0020]    In the third aspect, the invention provides a pharmaceutical composition comprising a safe and effective amount of DSPP and/or DSP protein and a pharmaceutically acceptable carrier. Preferably, said pharmaceutical composition is injection.  
           [0021]    In the fourth aspect, the invention provides a kit for detecting DGI-II and/or DGI-II with deafness comprising the primers which specifically amplify the DSPP gene or transcript. Preferably, the kit further comprises a probe that binds to the site of mutation.  
           [0022]    In view of the technical teaching of the invention, the other aspects of the invention will be apparent to the skilled in the art. 
       
    
    
     DESCRIPTION OF DRAWINGS  
       [0023]    [0023]FIG. 1 shows the gene structure of DSPP. This gene contains 5 exons and 4 introns. The full length is 8210 bp. Exon 1 (7-98), Exon 2 (2359-2437), Exon 3 (3577-3660), Exon 4 (3794-4780) and Exon 5 (5257-8201) encode DSPP. Exons 1-4 and part of Exon 5 (5257-5520) encode DSP, while another part of Exon 5 (5521-7893) encodes DPP.  
         [0024]    [0024]FIG. 2 shows the haplotype construction of STRP markers in 4q21 region in a dentinogenesis imperfecta type II family.  
         [0025]    [0025]FIG. 3 shows the haplotype construction of STRP markers in 4q21 region in a DGI-II with deafness family.  
         [0026]    [0026]FIGS. 4A and 4B show mutations in DSPP gene. FIG. 4A shows G1→T1 in position 1 of Exon 3, which causes codon GTT change into TTT, resulting in a corresponding amine acid change of Val→Phe. FIG. 4B shows G1→A1 in position 1 of Intron 3, which causes the mutation of splicing site. 
     
    
     DETAILED DESCRIPTION OF INVENTION  
       [0027]    After studying for several years, the inventors of the invention have, for the first time found and proved dentin sialophosphoprotein (DSPP) and/or dentin sialoprotein (DSP) have a close relationship with dentinogenesis imperfecta type II (DGI-II). In addition, the new function of DSPP/DSP was found, i.e., the changes of DSPP or DSP will cause DGI-II directly. Based on this discovery, the inventors accomplished the invention.  
         [0028]    Firstly, the inventors collected two genetic families affected by dentinogenesis imperfecta or dentinogenesis imperfecta with progressive hearing loss in China. Then they localized the disease gene of dentinogenesis imperfecta to the 4q21-22 region in Chromosome 4 by genotyping and linkage analysis with microsatellite markers Then, the inventors identified the candidate genes by the following steps:  
         [0029]    (1) Finding all of the genes mapped in 4q21-22 region, i.e., making the transcription map in 4q21-22 region;  
         [0030]    (2) Checking the expression situation of all of the genes in 4q21-22 region;  
         [0031]    (3) Determining the genes mapped in 4q21-22 region and expressed in dental pulp as the candidates for dentinogenesis imperfecta.  
         [0032]    The results showed that the candidate genes included DMP1, IBSP, SPP1, DSP, DPP and DSPP.  
         [0033]    Further, the inventors used PCR-SSCP technique to screen all candidate genes for mutation and found that the mutations in DSPP have a direct causality with dentinogenesis imperfecta, while other genes do not.  
         [0034]    Finally, the mode and site of DSPP mutation in two genetic families were identified by sequence analysis. In DGI-II family, sequencing revealed a G1→T1 mutation at position 1 of Exon 3 (position 3577 in SEQ ID NO:1). The mutation results in not only an amino acid change, but also a splicing site change which may cause the expression of intron, termination of translation in advance or frame shifting (FIG. 4A). Therefore, the normal DSPP (or DSP) protein is unable to be expressed.  
         [0035]    In another DGI-II with deafness family, the mutation is a G1→A1 mutation in position 1 of Intron 3 (position 3661 of SEQ ID NO:1). The mutation was predicted to result in splicing site change, which may cause the expression of intron, termination of translation in advance or frame shifting (FIG. 4B). Therefore, the normal DSPP (or DSP) protein is unable to be expressed. Further, it may influence the translation of signal peptide so that DSPP can not be correctly localized. Surprisingly, this mutation causes the patient affected with both DGI-II and deafness, suggesting that DSPP mutation is associated with deafness. It is possible to diagnose deafness, especially DGI-II with deafness, by detecting whether DSPP is normal or not.  
         [0036]    On the basis of this invention, one can design and exploit new drugs based on DSPP gene and its products (e.g., transcripts and proteins) as well as the interacting molecule. In addition, one can use DSPP gene in vitro to reconstruct teeth or remodel some tooth structure, such as dentin.  
         [0037]    Human DSPP mutation causes human dentinogenesis imperfecta type II. Based on the DSPP gene and its expression products, one can develop new drugs and diagnosis/treatment techniques for detecting and treating human DGI-II.  
         [0038]    Human DSPP Gene and Protein  
         [0039]    The detailed sequences of human DSPP gene and protein are available in Genbank (The accession number is AF163151) and some references, such as Gu, K., Chang, S., Ritchie, H. H., Clarkson, B. H. and Rutherford, R. B., Eur. J. Oral Sci. 2000 Feb: 108 (1):35-42. In Sequence Listing, human DSPP nucleotide sequence and amino acid sequence are shown in SEQ ID NO:1 and SEQ ID NO: 2, respectively. FIG. 1 shows the introns and exons of human DSPP.  
                                       Exon 1   7-98       mRNA   join (7-98, 2359-2437, 3577-3660, 3794-4780,           5257-8201)       Exon 2   2359-2437       CDS   join (2387-2437, 3577-3660, 3794-4780, 5257-7896)       sig_peptide   2387-2431       mat_peptide   join (2432-2437, 3577-3660, 3794-4780, 5257-7893)/           Product “DSPP”       mat_peptide   join (2432-2437, 3577-3660, 3794-4780, 5257-5520)/           Product “DSP”       Exon 3   3577-3660       Exon 4   3794-4780       Exon 5   5257-8201       mat_peptide   5521-7893       misc_feature   5596-5604 /note = “Region: cell binding domain”       PolyA_signal   7988-7993       PolyA_signal   8171-8176                  
 
         [0040]    The DSPP and/or DSP protein or polypeptide have various uses including but not limited to: curing disorders caused by low or no activity of DSPP and/or DSP protein (using directly as a medicine), and screening out antibodies, polypeptides or ligands which promote the function of DSPP and/or DSP. The expressed recombinant DSPP and/or DSP protein can be used to screen polypeptide library to find therapeutically valuable polypeptide molecules which activate the function of DSPP and/or DSP protein.  
         [0041]    In another aspect, the invention also includes polyclonal and monoclonal antibodies, preferably monoclonal antibodies, which are specific for polypeptides encoded by human DSPP DNA or fragments thereof. By “specificity”, it is meant an antibody that binds to the human DSPP gene products or fragments thereof. Preferably, the antibody binds to the human DSPP gene products or fragments thereof and does not substantially recognize nor bind to other antigenically unrelated molecules. Antibodies that bind to human DSPP and block human DSPP protein and those which do not affect the human DSPP function are included in the invention.  
         [0042]    The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically-active antibody fragments, e.g., a Fab′ or (Fab) 2  fragment, an antibody heavy chain, an antibody light chain, a genetically engineered single chain Fv molecule (Lander, et al., U.S. Pat. No. 4,946,778), or a chimeric antibody, e.g., an antibody which contains the binding specificity of a murine antibody, but the remaining portion of which is of human origin.  
         [0043]    The antibodies in the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human DSPP gene products, or its antigenic fragments can be administrated to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human DSPP or its antigenic fragments can be used to immunize animals to produce antibodies. The antibodies of the invention can be monoclonal antibodies which can be prepared by using hybridoma technique (See Kohler, et al., Nature, 256; 495,1975; Kohler, et al., Eur. J. Immunol. 6: 511,1976; Kohler, et al., Eur. J. Immunol 6: 292, 1976; Hammerling, et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981). Antibodies of the invention comprise those which block human DSPP function and those which do not affect human DSPP function. Antibodies in the invention can be produced by routine immunology techniques and using fragments or functional regions of human DSPP gene product. These fragments and functional regions can be prepared by recombinant methods or synthesized by a polypeptide synthesizer. The antibodies binding to unmodified human DSPP gene product can be produced by immunizing animals with gene products produced by prokaryotic cells (e.g.,  E. coli ), and the antibodies binding to post translationally modified forms thereof (e.g., glycosylated or phosphorylated polypeptide) can be acquired by immunizing animals with gene products produced by eukaryotic cells (e.g., yeast or insect cells).  
         [0044]    The antibody against human DSPP and/or DSP protein can be used in immunohistochemical method to detect the presence of DSPP and/or DSP protein in the biopsy specimen.  
         [0045]    The polyclonal antibodies can be prepared by immunizing animals, such as rabbit, mouse, and rat, with human DSPP and/or DSP protein. Various adjuvants, e.g., Freund&#39;s adjuvant, can be used to enhance the immunization.  
         [0046]    The substances that act with DSPP and/or DSP protein, e.g., inhibitors, agonists and antagonists, can be screened out by various conventional techniques, using the protein of the invention.  
         [0047]    The protein, antibody, inhibitor, agonist or antagonist of the invention provides different effects when administrated in therapy. Usually, these substances are formulated with a non-toxic, inert and pharmaceutically acceptable aqueous carrier. The pH typically ranges from 5 to 8, preferably from about 6 to 8, although pH may alter according to the property of the formulated substances and the diseases to be treated. The formulated pharmaceutical composition is administrated in conventional routine including, but not limited to, intramuscular, intravenous, subcutaneous, or topical administration. The topical administration at periodontal tissues is preferred.  
         [0048]    The normal DSPP and/or DSP can be directly used for curing disorders, e.g., DGI-II. The DSPP and/or DSP protein of the invention can be administrated in combination with other medicaments for DGI-II.  
         [0049]    The invention also provides a pharmaceutical composition comprising safe and effective amount of DSPP and/or DSP protein in combination with a suitable pharmaceutical carrier. Such a carrier includes but is not limited to saline, buffer solution, glucose, water, glycerin, ethanol, or the combination thereof. The pharmaceutical formulation should be suitable for the delivery method. The pharmaceutical composition of the invention may be in the form of injections which are made by conventional methods, using physiological saline or other aqueous solution containing glucose or auxiliary substances. The pharmaceutical compositions in the form of tablet or capsule may be prepared by routine methods. The pharmaceutical compositions, e.g., injections, solutions, tablets, and capsules, should be manufactured under sterile conditions. The active ingredient is administrated in therapeutically effective amount, e.g., from about lug to 5 mg per kg body weight per day. Moreover, the polypeptide of the invention can be administrated together with other therapeutic agents.  
         [0050]    When using pharmaceutical composition, the safe and effective amount of the DSPP and/or DSP protein or its antagonist or agonist is administrated to mammals. Typically, the safe and effective amount is at least about 0.1 ug/kg body weight and less than about 10 mg/kg body weight in most cases, and preferably about 0.1-100 ug/kg body weight. Of course, the precise amount will depend upon various factors, such as delivery methods, the subject health, and the like, and is within the judgment of the skilled clinician.  
         [0051]    The human DSPP and/or DSP polynucleotides also have many therapeutic applications. Gene therapy technology can be used in the therapy of abnormal cell proliferation, development or metabolism, which is caused by the loss of DSPP and/or DSP expression or the expression of abnormal or non-active DSPP and/or DSP. The methods for constructing a recombinant virus vector harboring DSPP and/or DSP gene are described in the literature (Sambrook, et al.). In addition, the recombinant DSPP and/or DSP gene can be packed into liposome and then transferred into the cells.  
         [0052]    The methods for introducing the polynucleotides into tissues or cells include: directly injecting the polynucleotides into tissue in the body, in vitro introducing the polynucleotides into cells with vectors, such as virus, phage, or plasmid, and then transplanting the cells into the body.  
         [0053]    The invention further provides diagnostic assays for quantitative and in situ measurement of DSPP and/or DSP protein level. These assays are well known in the art and include FISH assay and radioimmunoassay. The level of DSPP and/or DSP protein detected in the assay can be used to illustrate the importance of DSPP and/or DSP protein in diseases and to determine the diseases associated with DSPP and/or DSP protein.  
         [0054]    A method of detecting the presence of DSPP and/or DSP protein in a sample by utilizing the antibody specifically against DSPP and/or DSP protein comprises the steps of: contacting the sample with the antibody specifically against DSPP and/or DSP protein; observing the formation of antibody complex which indicates the presence of DSPP and/or DSP protein in a sample.  
         [0055]    The polynucleotide encoding DSPP and/or DSP protein can be used in the diagnosis and treatment of DSPP and/or DSP protein related diseases. In respect of diagnosis, the polynucleotide encoding DSPP and/or DSP can be used to detect whether DSPP and/or DSP is expressed or not, and whether the expression of DSPP and/or DSP is normal or abnormal, e.g., in the case of diseases. DSPP DNA sequences can be used in the hybridization with biopsy samples to determine the expression of DSPP. The hybridization methods include Southern blotting, Northern blotting and in situ blotting, etc., which are public and sophisticated techniques. The corresponding kits are commercially available. A part of or all of the polynucleotides of the invention can be used as probe and fixed on a microarray or DNA chip for analyzing the differential expression of genes in tissues and for the diagnosis of genes. The DSPP and/or DSP specific primers can be used in RNA-polymerase chain reaction and in vitro amplification to detect the transcripts of DSPP and/or DSP.  
         [0056]    Further, detection of the mutation of DSPP and/or DSP gene is useful for the diagnosis of DSPP and/or DSP protein related diseases. The mutation forms of DSPP and/or DSP include site mutation, translocation, deletion, rearrangement and any other mutations compared with the normal wild-type DSPP and/or DSP DNA sequence. The conventional methods, such as Southern blotting, DNA sequencing, PCR and in situ blotting, can be used to detect mutation. Moreover, mutation sometimes affects the expression of protein. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether the gene is mutated or not.  
         [0057]    The invention is further illustrated by the following examples. It is appreciated that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples, they are performed under routine conditions, e.g., those described by Sambrook et al., in Molecule Clone: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturers, unless otherwise specified.  
       EXAMPLE 1  
       [0058]    DGI-II family had 42 members, and DGI-II with deafness family had 14 members. All individuals were subjected to careful clinical examination and recorded in details by experienced dentists. The patients with deafness were examined carefully by otologists and identified by pure tone audiogram and brain stem evoked potential. 5 ml blood samples in the families were collected by standard venipuncture and stocked by ACD solution. DNA was extracted using the following method:  
         [0059]    Preparation of Blood DNA Sample  
         [0060]    Blood DNA samples were extracted by Qiagen kit according to manufacturer&#39;s instructions. The steps were as follows:  
         [0061]    a. Add 20 ul Proteinase K, 200 ul blood sample and 200 ul Buffer AL into a 1.5 ml microcentrifuge tube. Mix by pulse-vortexing for 15 seconds.  
         [0062]    b. Incubate for digestion at 56° C. for 10 minutes. Add 210 ul 100% ethanol to the sample, and briefly centrifuge for 10 seconds.  
         [0063]    c. Carefully apply the mixture onto a QIAamp spin column and centrifuge at 8000 rpm for 1 minute.  
         [0064]    d. Discard the filtrate and transfer the QIAamp spin column in another 2 ml collection tube.  
         [0065]    e. Add 500 ul Buffer AW1 into QIAamp spin column, centrifuge at 8000 rpm for 1 minute.  
         [0066]    f. Discard the filtrate and add 500 ul Buffer AW2 into QIAamp spin column, centrifuge at 14000 rpm for 3 minutes.  
         [0067]    g. Discard the filtrate and place the QIAamp spin column in a new 1.5 ml microcentrifuge tube.  
         [0068]    h. Add 200 ul Buffer AE into QIAamp spin column, incubate at room temperature for 5 minutes, and centrifuge at 8000 rpm for 1 minute. The filtrate collected in the tube was DNA solution from blood sample.  
         [0069]    i. DNA quality was determined by 1% agarose gel electrophoresis. The DNA quantity was determined by UV spectrophotometer. The DNA samples were stored at −20° C.  
       EXAMPLE 2  
       [0070]    1 Genotyping:  
         [0071]    The sequences of high polymorphic STR markers in region 4q21 were obtained from Genome Database and markers A-G were D4S2691, D4S1534, GATA62 μl 1, DSP, DMP1, SPP1, D4S451, respectively. PCR amplifications were carried out following LI-COR company manual for the touchdown program and using PTC-225 DNA Engine Tetrad (MJ-Research Inc.). PCR reactions were in 10 ul system containing 20 ng genomic DNA template, 2.0 mM dNTP, 1.0 pmol M13-tailed forward primer and reverse primer, 1.0 pmol fluorescent M13 primer, 1.5 mM MgCl 2 , 10 mM Tris-HCl, and 1U AmpliTaq Gold Taq Polymerase (Perkin-Elmer Corp.). The reaction system was initially denatured at 95° C. for 8 minutes, followed by 4 cycles of denaturing at 95° C. for 45 seconds, annealing at 68° C. for 2 minutes with a drop of 2° C. per cycle until 60° C., and extending at 72° C. for 1 minute, and by a second set of 2-4 cycles of denaturing at 95° C. for 45 seconds, annealing at 58° C. for 1 minute with a drop of 2° C. per cycle until 50-54° C., and extending at 72° C. for 1 minute, and then by 20-30 cycles of denaturing at 95° C. for 30 seconds, annealing at 50-54° C. for 30 seconds and extending at 72° C. for 30 seconds. Finally the samples were extended at 72° C. for 15 minutes. PCR products and fluorescent-labeled standard size DNA markers were electrophoresed on a LI-COR automated sequencer on a polyacrylamide gel. Data were collected and analyzed by Base Image 4.1 and Gene Image 3.12 software, while linkage ready pedigree files were generated. These files were used for linkage analysis and haplotype analysis.  
         [0072]    2. Linkage Analysis and Haplotype Analysis  
         [0073]    DGI-II hereditary locus was modeled as an autosomal dominant inheritance with 100% penetrance in a two-allele system. The frequency of disease gene was set to 0.0001, the frequencies of STRs were assumed to be uniformly distributed. Two-point linkage analysis was performed by using MLINK and ILINK program from the LINKAGE version 5.10 software package. Haplotype construction was performed using SIMWALK2 version 2.31 and Cyrillic version 2.02 software.  
         [0074]    The pedigree data are shown in Tables 1-2 and FIGS.  2 - 3 .  
                                                                                                                           TABLE 1                           Disease locus in DGI-II pedigree and STRP two-point linkage analysis in       4q21 region            Loca-                tion   Lod score at θ   Maximum            marker   0.0   0.01   0.05   0.1   0.2   0.3   0.4   Lod   θ                    A   −∞   −0.11   2.19   2.76   2.59   1.81   0.83   2.76   0.1       B   1.65   1.62   1.51   1.37   1.09   0.78   0.42   1.65   0.0       C   7.63   7.50   6.96   6.25   4.74   3.09   1.36   7.63   0.0       D   6.06   5.96   5.53   4.98   3.82   2.57   1.24   6.06   0.0       E   8.24   8.11   7.54   6.80   5.22   3.49   1.67   8.24   0.0       F   8.38   8.24   7.67   6.93   5.32   3.55   1.64   8.38   0.0       G   7.34   7.23   6.77   6.16   4.87   3.44   1.84   7.34   0.0                  
 
         [0075]    [0075]                                                                                                                           TABLE 2                           Disease locus in DGI-II with deafness pedigree and Lod score in 4q21       region            Loca-                tion   Lod score at θ   Maximum            marker   0.0   0.01   0.05   0.1   0.2   0.3   0.4   Lod   θ                    A   −∞   −2.86   −1.48   −0.92   −0.42   −0.18   −0.05   −0.05   0.4       B   −∞   0.67   1.19   1.25   1.04   0.65   0.21   1.25   0.1       C   1.20   1.8   1.07   0.93   0.63   0.33   0.08   1.20   0.0       D   −0.14   −0.09   −0.05   −0.02   −0.00   −0.00   −0.00   −0.00   0.2       E   0.91   0.92   0.91   0.86   0.67   0.41   0.14   0.92   0.01       F   2.71   2.66   2.46   2.21   1.65   1.02   0.37   2.71   0.0       G   2.11   2.07   1.91   1.70   1.24   0.73   0.23   2.07   0.0                    
         [0076]    The results suggested that the disease genes in DGI-II and DGI-II with deafness pedigrees were linked with STRP markers in 4q21 region.  
       EXAMPLE 3  
       [0077]    Mutation Screening of Candidate Genes  
         [0078]    Using Primer 5.0 software (http://www/PrimerBiosoft.com), we designed primers to amplify exons and the splice junctions between exons and introns of DSP gene (Table 3). PCR-SSCP technique was used to screen DSP gene for mutation. PCR products were electrophoresed on 10% polyacrylamide gel and 9.3% polyacrylamide gel with 4% glycerol. Then, the gels were silver stained according to standard protocol.  
         [0079]    Primers were as follows:  
                                         TABLE 3                           Primer Sequences in DSPP Coding Region                Primer                       Name   Sequence   No.   bp               DSPP-E1 F   5′-TGCAAAAGTCCATGACAGTG-3′   SEQ ID   128                   NO:3               DSPP-E1 R   5′-TCAGTTGGTTCTGAGTAAAAAGGA-3′   SEQ ID               NO:4               DSPP-E2 F   5′-AAGTAATTTTGTGCTGTTCCTTT-3′   SEQ ID   149               NO:5               DSPP-E2 R   5′-AACAAAGTGAAGAGGTTTTCT-3′   SEQ ID               NO:6               DSPP-E3 F   5′-AAGAACCTTTTCAATTGCTAGT-3′   SEQ ID   189               NO:7               DSPP-E3 R   5′-TGGAGAAGTTAATGGAATGTAGCA-3′   SEQ ID               NO:8               DSPP-E4 F   5′-TGCAATTTGCTTTCCTTCAA-3′   SEQ ID   205               NO:9               DSPP-E4 R   5′-CCTCTTCGTTTGCTAATGTGG-3′   SEQ ID               NO:10               DSPP-E5 F   5′-TCACAAGGTAGAAGGGAATG-3′   SEQ ID   226               NO:11               DSPP-E5 R   5′-GTTTGTGGCTCCAGCATTGT-3′   SEQ ID               NO:12               DSPP-E6 F   5′-GGGACACAGGAAAAGCAGAA-3′   SEQ ID   243               NO:13               DSPP-E6 R   5′-TGTTATTGCTTCCAGCTACTTGAG-3′   SEQ ID               NO:14               DSPP-E7 F   5′-CAATGAGGATGTCGCTGTTG-3′   SEQ ID   206               NO:15               DSPP-E7 R   5′-TATCCAGGCCAGCATCTTCT-3′   SEQ ID               NO:16               DSPP-E8 F   5′-CACCTCAGATCAACAGCAAGAG-3′   SEQ ID   226               NO:17               DSPP-E8 R   5′-TCTTCTTTCCCATGGTCCTG-3′   SEQ ID               NO:18               DSPP-E9 F   5′-ATGAAGAAGCAGGGAATGGA-3′   SEQ ID   232               NO:19               DSPP-E9 R   5′-ATTCTTTGGCTGCCATTGTC-3′   SEQ ID               NO:20               DSPP-E10 F   5′-TGATGGAGACAAGACCTCCAA-3′   SEQ ID   205               NO:21               DSPP-E10 R   5′-TGCCATTGAAAGAAATCAGC-3′   SEQ ID               NO:22               DSPP-E11 F   5′-TTCTTTCCTCCATCCTTCCA-3′   SEQ ID   194               NO:23               DSPP-E11 R   5′-TTCTGATTTTTGGCCAGGTC-3′   SEQ ID               NO:24               DSPP-E12 F   5′-GGCAATGTCAAGACACAAGG-3′   SEQ ID   236               NO:25               DSPP-E12 R   5′-TCTCCTCGGCTACTGCTGTT-3′   SEQ ID               NO:26               DSPP-E13 F   5′-TGCAAGGAGATGATCCCAAT-3′   SEQ ID   231               NO:27               DSPP-E13 R   5′-TGTCATCATTCCCATTGTTACC-3′   SEQ ID               NO:28               DSPP-E14 F   5′-CAAAAGGAGCAGAAGATGATGA-3′   SEQ ID   243               NO:29               DSPP-E14 R   5′-TGCTGTCACTGTCACTGCTG-3′   SEQ ID               NO:30               DSPP-E15 F   5′-GCAGTGATAGTAGTGACAGCAGTG-3′   SEQ ID   205               NO:31               DSPP-E15 R   5′-TTGCTGCTGTCTGACTTGCT-3′   SEQ ID               NO:32               DSPP-E16 F   5′-CAAATCAGACAGTGGCAAAGG-3′   SEQ ID   508               NO:33               DSPP-E16 R   5′-GCTCTCACTGCTATTGCTGCT-3′   SEQ ID               NO:34               DSPP-E17 F   5′-GCAAGTCAGACAGCAGCAAA-3′   SEQ ID   598               NO:35               DSPP-E17 R   5′-CTGCTGTCGCTATCACTGCT-3′   SEQ ID               NO:36               DSPP-E18 F   5′-ATAGCAACGACAGCAGCAAT-3′   SEQ ID   583               NO:37               DSPP-E18 R   5′-TCGCTGCTATTGCTATCACTG-3′   SEQ ID               NO:38               DSPP-E19 F   5′-GCAACAGCAGTGATAGTGACA-3′   SEQ ID   598               NO:39               DSPP-E19 R   5′-CTGCTGTCGCTGCTTTCA-3′   SEQ ID               NO:40               DSPP-E20 F   5′-AGCAGCGACAGCAGTGATAT-3′   SEQ ID   500               NO:41               DSPP-E20 R   5′-TTGTTACCGTTACCAGACTTGC-3′   SEQ ID               NO:42               DSPP-E21 F   5′-TGACAGCACATCTGACAGCA-3′   SEQ ID   261               NO:43               DSPP-E21 R   5′-TCCCCCAGTTGTTTTTGTTT-3′   SEQ ID               NO:44                  
 
         [0080]    PCR products were sequenced to determine the type and location of mutations.  
         [0081]    1. The DNA fragments that showed a changed electrophoresis pattern in SSCP analysis were amplified by standard PCR.  
         [0082]    2. PCR products were purified with Millipore spin column.  
         [0083]    3. Sequencing Reaction:  
                                                           (1)   Reaction system                   Reaction mixture   2 ul               Primers (0.8 mM)   2 ul               Purified PCR products   3 ul           (2)   Reaction conditions:               96° C.   30 sec               96° C.   30 sec               50° C.    5 sec               60° C.    4 min               60° C.    4 min                      
 
         [0084]    Total 35 cycles  
         [0085]    (3) Precipitation of the Product of Sequencing Reaction  
         [0086]    Add 9 volumes of 70% ethanol into the sequencing product, incubate at 4° C. for 3 minutes.  
         [0087]    Centrifuge at 4° C. at 4000 rpm for 30 minutes.  
         [0088]    Place the centrifuge tube upside down and continue to centrifuge until the speed reaches 1300 rpm at 4° C.  
         [0089]    (4) Loading and Sequencing Samples  
         [0090]    Add 2 ul Loading Dye buffer into precipitated products of sequencing reaction.  
         [0091]    Incubate at 90° C. for 2 minutes and place it on ice immediately.  
         [0092]    Load samples into ABI PRISM automated DNA sequencer to sequence.  
         [0093]    The sequencing results were shown in FIGS. 4A and 4B. In DGI-II family, sequencing revealed a G1→T1 mutation at position 1 of Exon 3 (position 3577 in SEQ ID NO:1). This mutation resulted in not only an amino acid change, but also a splicing site change that might cause the expression of intron, termination of translation in advance or frame shifting (FIG. 4A). Therefore, the normal DSPP (or DSP) protein was unable to be expressed.  
         [0094]    In another DGI-II with deafness family, the mutation was a G1→A1 mutation in position 1 of Intron 3 (position 3661 of SEQ ID NO:1). This mutation was predicted to result in splicing site change which may cause the expression of intron, termination of translation in advance or frame shifting (FIG. 4B). Therefore, the normal DSPP (or DSP) protein was unable to be expressed. Further, it might influence the translation of signal peptide so that DSPP could not be correctly localized. Surprisingly, this mutation caused the patient affected with both DGI-II and deafness, suggesting that DSPP mutation was associated with deafness. It is possible to diagnose deafness, especially DGI-II with deafness, by detecting whether DSPP is normal or not.  
         [0095]    Discussion  
         [0096]    1. Linkage and Haplotype Analysis  
         [0097]    We used seven STR markers in 4q21 region to genotype DGI-II and DGI-II with deafness families. Linkage and haplotype construction showed that the disease gene in DGI-II family was linked with 4q21 and the maximum LOD score was 8.38 at SPP1 locus (θ=0.00) (Table 1, FIG. 2) and the disease gene in DGI-II with deafness was also linked with STR markers in 4q21 region and the maximum LOD score was 2.71 (0=0.00) (Table 2, FIG. 3).  
         [0098]    2. Mutation Screening of Candidate Genes and Confirmation by Sequencing  
         [0099]    We designed 22 primers overlapping the DSPP gene to screen for mutations and identify mutations by sequencing. We found the disease gene in DGI-II family was linked with the STR markers in 4q21 region, while the disease gene in DGI-II with deafness was also linked with STR markers in this region. These mutations were not observed in 100 normal and unaffected individuals. It suggests that these mutations should be the cause of DGI-II disease.  
         [0100]    DPP and DSP are two small polypeptides which have specific chemical properties and are cleaved from a single transcripts of DSPP gene. Both of them are expressed specifically in dental pulp tissue and may also be expressed in cochleae. DSP is a Glu-, Ser- and Gly-rich protein with many phosphorylation sites, which are predicted to be involved in dentin mineralization. DPP affects mineralization in two ways. Low concentration of DPP protein is able to bind to interspace of collagen I and initiate formation of phosphorum apatite crystals, while high concentration of DPP protein binds to the growing crystals, affects the size and form of crystals, and decreases the growth of crystals. It is necessary to further study the mechanism that the mutations in DSPP gene cause dentinogenesis imperfecta and deafness.  
         [0101]    All the documents cited herein are incorporated into the invention as reference, as if each of them is individually incorporated. Further, it would be appreciated that, in the above teaching of the invention, the skilled in the art could make certain changes or modifications to the invention, and these equivalents would still be within the scope of the invention defined by the appended claims of the present application.  
       REFERENCES  
       [0102]    1. Witkop C J et al. Hereditary defects in enamel and dentin. Acta Genet 1957;7:236˜239  
         [0103]    2. Cetta G et al. Third international conference on osteogenesis imperfecta. Ann NY Avad Sci, 1998  
         [0104]    3. Takagi Y et al. Matrix protein difference between human normal and dentinogenesis imperfecta dentin. In the chemistry and biology of mineralized connective tissues. Veis A, editor, New York: Elsevier/North-Holand. 1981  
         [0105]    4. Witkop C J, et al. Medical and dental findings in the Brandywine isolate. AL J Med Sci 1966;3:382˜403  
         [0106]    5. Bixler D, et al. Dentinogenesis imperfecta: genetic variation in a six-generation family. J. Dent. Res. 1968;48:1196˜1199  
         [0107]    6. Mikkelsen, M et al. Possible localization of Gc-system on chromosome 4. Loss of long arm 4 material associated with father-child incompatibility within the Gc-system. Hum. Hered. 1988;27: 105˜107  
         [0108]    7. Ball. S P, et al. Linkage between dentinogenesis imperfecta and Gc. Ann. Hum. Genet. 1982;46:35˜40  
         [0109]    8. Crall M G. Genetic marker study of dentinogenesis imperfecta. Proc Finn Dent Soc. 1992;88:285˜293  
         [0110]    9. Crodby A H, et al. Genetic mapping of dentinogenesis imperfecta type II Locus. Am. J. Hm. Genet. 1995;57:832˜839  
         [0111]    10. Aplin H. M, et al. Refinement of the dentinogenesis imperfecta type II locus to an interval of less than 2 centimorgans at chromosome 4q21 and the creation of a yeast artificial chromosome contig of the critical region. J. Dent. Res. 1999;78(6):1270˜1276  
         [0112]    [0112] 
     
       
       
         1 
         
           
             44  
           
           
             1  
             8201  
             DNA  
             Homo sapiens  
           
            1 

attgtcatgc aaaagtccag gacagtgggc cactttcagt cttcaaagag aaagataaga     60 

aattctggat tttcaaaatc cttttgaagc cttttaaggt aagatgaaat atccttttta    120 

ctcagaacca actgattcat ttagaaagaa ctttgaattt caaagatgaa gccagtttga    180 

ttttaagaag cgagtacccc ttaatgatta gattgtatgc ttcctttttg acttgtcata    240 

ttgatagtat gtataaaaga taacggacga ttacgaccta aggaagagat agattgggaa    300 

gaagaaagac ctcgtactga aaaattggcc aactgaggtg gaaatttgac aattaactat    360 

ctgggcactt tgattagttt tgataaaaaa tgagataact cagatttcaa aaatccacct    420 

tgggctttca aacaaggctt caattaggct ttgcttttta gtattttatt acttactatt    480 

acttattatt tattgtccca catgaaatga aatttagcaa tcactaatga tgccaaatct    540 

aattgctaaa tgaaatgaag ctaaatctca tttcattagt aacaataaat gaaataatct    600 

gatggagctt cacaaattct gaagtctttg tttcatgctg aggtcacctg ggccattttt    660 

attgtagtct tcgaagtcat tcacctgcct tggaaacggt gataaccatc atggaattgt    720 

tcaggagtgg agctgaaaga gagatgtagt ggtcagattt ctgaactgta gctcagaaac    780 

tggacacgta tcactctggc cttggctgca ggtacctttc cagtatgctg aggctcttcc    840 

aaatcacagt gcagacgggc cttctgcaga gctatgtaat gattaggctt gggactgcaa    900 

agtacaggat aactgtggct tagtaaacag ctggccttca acatctgtgc cccagagctc    960 

tgcatgatac ttgtcctggt gtcacctcag cctcacttga atctatggca tttcagaagg   1020 

agctctagct gttcttggct ttctgttgaa cagctataag aatgagcact tttttccctc   1080 

tcagtagctc tggaactgtg tcatctctcc tgtgagaaaa cgccagtaat tctcatgaca   1140 

gttgatattc agtgaagttt tattatattt tcactaccac cattaaattc aatcaaagcc   1200 

attttatgac atgcagcatt ataatctata catctggtgg gagttcatga aataggagta   1260 

aaactctcct ttctatcatt acttcaagaa atccaacttg caatataaat taattttttt   1320 

actcacacag attataaaat gtctattcca acttatcaga aacatgtttt agaccatttc   1380 

tgaatttgaa ttctaacagg gatgaagaat catgatttta gaagtcccat aaaataattg   1440 

ctatcattta ttcaaaaatt gcaaagtgcc tgaagcaatg ctagatattg ctgatagtca   1500 

taaatattta tcaacaacat tcagaaaacg tttttttctg tgctttgcat tggaatacaa   1560 

taatcaccaa gacactctcc tgggcctcag gagcttacag gaaatcaggg caacacataa   1620 

gtaactaggc aattttaaac agtgcaatgc gttaccagtg agacgtgcaa acttccttgg   1680 

tataaaaagg aaagagatac caaataccct ttgaagtggc gtcagagagg gcgtctcaga   1740 

gataattcta ccaaacttca ggataatcct gaggtgcagg tgttgttatt attccaggtg   1800 

gagggataat aaacctactt aaatttctca agcttacaca gcaagtagca ggggtaacat   1860 

ttgaacccag gtctctgaat acaaaccccg tattctttcc actagcgtag gctccctcat   1920 

gttagtaatt tctttctctt aaagtctggt atagctcaat tctatagatt tggagtaagg   1980 

atgacaagtg ttttaccttt gaagcacaat ttcagcagaa ttagttagta cttgattaaa   2040 

gctattcaga agagaaatag atgtttttac acccaagaat tgcagaagaa caaagttaca   2100 

gctatgccct ttgtacctat tatggtgttt tccttcattg gcacaggcag aaaaaaatct   2160 

aggaagctac attagtgctg agcctggtga tgtccccata accacaccag gtatgttctg   2220 

gaccatcgta tgtcttctcg tgttagatac atgcttcttg tccaggaaaa gggcaaatgc   2280 

ttacacatca aaataatata gtactatgat tttcccttta ctttataagt aattttgtgc   2340 

tgttcctttt ttatacagcc attgattatt attattccta aagaaaatga agataattac   2400 

atatttttgc atttgggcag tagcatgggc cattccagta agtatgcctt tcttagaaaa   2460 

cctcttcact ttgttatctt ttttaaccta acattaatac aaaatgtagt gtgtgtgtgt   2520 

gtgtgtgtgt gtgtgtgtgt gtgcatgtac atgtgtgtat atatgtgtgt gtgtatatat   2580 

gtttccttaa ttttttttaa caggctgagt ctaaacattt agatttgcac taagggcttt   2640 

atgtgatatc tgtgaggttt caacaaaacc actccaattc atcgtctcat tcctctatag   2700 

aaactcatat ctcgtctgaa ggattattat tatttaaaac atttattcag attaatttac   2760 

acttaatgcc cagaagtcat ggagactttg tccatctttg cttcatactc tgtgaatttc   2820 

attctaatac gaacaaagtc tgtgctgttt aggaagtttc caagaaagaa taataagaaa   2880 

aagtagattt tttttcaaca tataggagac taatttttca ctcagagtta ttatttatgt   2940 

gctcactgtg gaaaatttgg aatatatgac gaaaaccaat aaaaaattga gaaaattcaa   3000 

ccatttataa ttttactagc cagccatcat gtttaacatt ttcatatgct ttcataatac   3060 

caaacatttg gtatttatgt agttgaaaat gttctcaagt atttcaaatg tgctcttgca   3120 

gagcacagaa gtatactagc gtaatacttg attttgcttc tgtgcaggct ctggtcacgc   3180 

ctcctgttct cttaagagtt ttcatcagga ttacacttag agcgggtttg tgctagtgca   3240 

agaggctttt tgtagagaaa caccagaggt ctatcccctc gtctttctac aagactcttt   3300 

ccttctacag ttgagataag tgggctgatc taacacgtcc ataaaattgg taataccaca   3360 

gtgaaaaata tccatgtacc cagtttaaat tctacacaag ccctgtaaga agccacttct   3420 

cttttctatc tgattagatc atactttggc ctttgtgtta aacctttctt cttcatggag   3480 

ggaagaatat ttgtgtgtgt gtgtgtgtgt gtgcacgctc acacacatat tcacaaataa   3540 

gaaccttttc aatagccagt attttctact tggcaggttc ctcaaagcaa accactggag   3600 

agacatgtcg aaaaatccat gaatttgcat ctcctagcaa gatcaaatgt gtcagtacag   3660 

gtataggatg taatatattt cattttattt cctatttctg agttgctaca ttccattaac   3720 

ttctccaaga ttgcaatttg ctttccttca agatcattga cactcataat tgattgaatt   3780 

gtttcttttt caggatgagt taaatgccag tggaaccatc aaagaaagtg gtgtcctggt   3840 

gcatgaaggt gatagaggaa ggcaagagaa tacccaagat ggtcacaagg gagaagggaa   3900 

tggctctaag tgggcagaag taggagggaa gagtttttct acatattcca cattagcaaa   3960 

cgaagagggg aatattgagg gctggaatgg ggacacagga aaagcagaaa catatggtca   4020 

tgatggaata catgggaaag aagaaaacat cacagcaaat ggcatccagg gacaagtaag   4080 

catcattgac aatgctggag ccacaaacag aagcaacact aatggaaata ctgataagaa   4140 

tacccaaaat ggggatgttg gcgatgcagg tcacaatgag gatgtcgctg ttgtccaaga   4200 

agatggacct caagtagctg gaagcaataa cagtacagac aatgaggatg aaataattga   4260 

gaattcctgt agaaacgagg gtaatacaag tgaaataaca cctcagatca acagcaagag   4320 

aaatgggact aaggaagctg aggtaacacc aggcactgga gaagatgctg gcctggataa   4380 

ttccgatggg agtcctagtg ggaatggagc agatgaggat gaagacgagg gttctggtga   4440 

tgatgaagat gaagaagcag ggaatggaaa agacagtagt aataacagca agggccagga   4500 

gggccaggac catgggaaag aagatgatca tgatagtagc ataggtcaaa attcggatag   4560 

taaagaatat tatgaccctg aaggcaaaga agatccccat aatgaagttg atggagacaa   4620 

gacctccaag agtgaggaga attctgctgg tattccagaa gacaatggca gccaaagaat   4680 

agaggacacc cagaagctca accatagaga aagcaaacgc gtagaaaata gaatcaccaa   4740 

agaatcagag acacatgctg ttgggaagag ccaagataag gttagtttgt aaagctgatt   4800 

tctttcaatg gcagtttaaa ttcttcccct ccatctattg atgctagcac aaaaataaac   4860 

catgacaagc atccatgtat ttttgtatcc atattacttg actatttaag gaaatctaga   4920 

gtccttacta gacttcgaga tagaacaact ttaaacatct tacatttctg ataacttagt   4980 

tataattcta gaaaagtctt atgtgaaatc atggatcccc atgtaattgt ttacaaaagt   5040 

tcctactggg taggaatgtg gatgaatttt taaggaatct aagcaccagg atgctttcaa   5100 

ttacagaata aagcacattt tcacaaataa ctgtgaagta ctagaaatgt aactcctatc   5160 

cctatggcaa cttttcccag ttattcttcc tcagatcaat gcaattttgc agcaaatatt   5220 

cactagttaa tcattctttc ctccatcctt ccatagggaa tagaaatcaa gggtcccagc   5280 

agtggcaaca gaaatattac caaagaagtt gggaaaggca acgaaggtaa agaggataaa   5340 

ggacaacatg gaatgatctt gggcaaaggc aatgtcaaga cacaaggaga ggttgtcaac   5400 

atagaaggac ctggccaaaa atcagaacca ggaaataaag ttggacacag caatacaggt   5460 

agtgacagca atagtgatgg atatgacagt tatgattttg atgataagtc catgcaagga   5520 

gatgatccca atagcagtga tgaatctaat ggcaatgatg atgctaattc agaaagtgac   5580 

aataacagca gtagccgagg agatgcttct tataactctg atgaatcaaa agataatggc   5640 

aatggcagtg actcaaaagg agcagaagat gatgacagtg atagcacatc agacactaat   5700 

aatagtgaca gtaatggcaa tggtaacaat gggaatgatg acaatgacaa atcagacagt   5760 

ggcaaaggta aatcagatag cagtgacagt gatagtagtg atagcagcaa tagcagtgat   5820 

agtagtgaca gcagtgacag tgacagcagt gatagcaaca gtagcagtga tagtgacagc   5880 

agtgacagtg acagcagtga tagcagtgac agtgatagta gtgatagcag caatagcagt   5940 

gacagtagtg acagcagtga tagcagtgac agtagtgata gtagtgacag cagtgacagc   6000 

aagtcagaca gcagcaaatc agagagcgac agcagtgata gtgacagtaa gtcagacagc   6060 

agtgacagca acagcagtga cagtagtgac aacagtgata gcagcgacag cagcaatagc   6120 

agtaacagca gtgatagtag tgacagcagt gatagcagtg acagcagcag tagcagtgac   6180 

agcagcagta gcagtgacag cagcaacagc agtgatagta gtgacagtag tgacagcagc   6240 

aatagcagtg agagcagtga tagtagtgac agcagtgata gtgacagcag tgatagtagt   6300 

gacagcagta atagtaacag cagcgatagt gacagcagca acagcagcga tagcagtgac   6360 

agcagtgata gcagtgacag cagcaacagc agtgacagta gcgatagcag tgacagcagc   6420 

aacagcagtg acagcagtga tagcagtgac agcagtgata gtagtgacag cagcaacagc   6480 

agtgatagca acgacagcag caatagcagt gacagcagtg atagcagcaa cagcagtgat   6540 

agcagcaaca gcagtgatag cagtgatagc agtgacagca gtgatagcga cagcagcaat   6600 

agcagtgaca gcagtaatag tagtgacagc agcgatagca gcaacagcag tgatagcagc   6660 

gacagcagcg atagcagtga cagcagtgat agcgacagca gcaatagaag tgacagtagt   6720 

aatagtagtg acagcagcga tagcagtgac agcagcaaca gcagtgacag cagtgatagt   6780 

agtgacagca gtgacagcaa cgaaagcagc aatagcagtg acagcagtga tagcagcaac   6840 

agcagtgata gtgacagcag tgatagcagc aacagcagtg acagcagtga tagcagcaac   6900 

agcagtgata gcagtgaaag cagtaatagt agtgacaaca gcaatagcag tgacagcagc   6960 

aacagcagtg acagcagtga tagcagtgac agcagtaata gtagtgacag cagcaatagc   7020 

ggtgacagca gcaacagcag tgacagcagt gatagcaata gcagcgacag cagtgacagc   7080 

agcaacagca gcgatagcag tgacagcagt gatagcagtg acagcagtga cagcagtgat   7140 

agcagcaaca gcagtgatag cagtgacagc agtgacagca gtgatagcag taatagtagt   7200 

gacagcagca acagcagtga cagcagcgat agcagtgaca gcagcgatag cagtgacagc   7260 

agtgacagca gcaatagcag tgacagcagt gacagcagcg acagcagtga tagcagtgac   7320 

agcagtggca gcagcgacag cagtgatagc agtgacagca gtgatagcag cgatagcagt   7380 

gacagcagcg acagcagtga cagcagtgac agcagtgaaa gcagcgacag cagcgatagc   7440 

agcgacagca gtgacagcag cgacagcagt gacagcagcg atagcagcga cagcagcgac   7500 

agcagcgata gcagtgacag cagcaatagc agtgatagca gcgacagcag tgatagcagt   7560 

gacagcagcg acagcagcga tagcagcgac agcagtgata gtagtgatag cagtgacagc   7620 

agtgacagca gcgacagcag tgacagcagc gacagcagtg acagcagcga cagcagtgac   7680 

agcaatgaaa gcagcgacag cagtgacagc agcgatagca gtgacagcag caacagcagt   7740 

gacagcagcg acagcagtga tagcagtgac agcacatctg acagcaatga tgagagtgac   7800 

agccagagca agtctggtaa cggtaacaac aatggaagtg acagtgacag tgacagtgaa   7860 

ggcagtgaca gtaaccactc aaccagtgat gattagaaca aaagaaaaac ccataagatt   7920 

ccttttgtga aaagtttggt aatgggatag gaaaaaaaga tttccaagaa agtaaagaaa   7980 

ggggagaaat aaacataaga cgtatgtaaa caaaaacaac tgggggaatc aaatcaaaca   8040 

gttggattca gaaccaagac ctaactcctg cagagacaga ctctgaatgc atgacctttg   8100 

gtacatgcct gttaatattc atgttctgaa aatattttgt taaaagtgta aatctaaaca   8160 

taaaagaaca attaaaatat tctttaatac ttcacacaga a                       8201 

 
           
             2  
             1253  
             PRT  
             Homo sapiens  
           
            2 

Met Lys Ile Ile Thr Tyr Phe Cys Ile Trp Ala Val Ala Trp Ala Ile 
1               5                   10                  15 

Pro Val Pro Gln Ser Lys Pro Leu Glu Arg His Val Glu Lys Ser Met 
            20                  25                  30 

Asn Leu His Leu Leu Ala Arg Ser Asn Val Ser Val Gln Asp Glu Leu 
        35                  40                  45 

Asn Ala Ser Gly Thr Ile Lys Glu Ser Gly Val Leu Val His Glu Gly 
    50                  55                  60 

Asp Arg Gly Arg Gln Glu Asn Thr Gln Asp Gly His Lys Gly Glu Gly 
65                  70                  75                  80 

Asn Gly Ser Lys Trp Ala Glu Val Gly Gly Lys Ser Phe Ser Thr Tyr 
                85                  90                  95 

Ser Thr Leu Ala Asn Glu Glu Gly Asn Ile Glu Gly Trp Asn Gly Asp 
            100                 105                 110 

Thr Gly Lys Ala Glu Thr Tyr Gly His Asp Gly Ile His Gly Lys Glu 
        115                 120                 125 

Glu Asn Ile Thr Ala Asn Gly Ile Gln Gly Gln Val Ser Ile Ile Asp 
    130                 135                 140 

Asn Ala Gly Ala Thr Asn Arg Ser Asn Thr Asn Gly Asn Thr Asp Lys 
145                 150                 155                 160 

Asn Thr Gln Asn Gly Asp Val Gly Asp Ala Gly His Asn Glu Asp Val 
                165                 170                 175 

Ala Val Val Gln Glu Asp Gly Pro Gln Val Ala Gly Ser Asn Asn Ser 
            180                 185                 190 

Thr Asp Asn Glu Asp Glu Ile Ile Glu Asn Ser Cys Arg Asn Glu Gly 
        195                 200                 205 

Asn Thr Ser Glu Ile Thr Pro Gln Ile Asn Ser Lys Arg Asn Gly Thr 
    210                 215                 220 

Lys Glu Ala Glu Val Thr Pro Gly Thr Gly Glu Asp Ala Gly Leu Asp 
225                 230                 235                 240 

Asn Ser Asp Gly Ser Pro Ser Gly Asn Gly Ala Asp Glu Asp Glu Asp 
                245                 250                 255 

Glu Gly Ser Gly Asp Asp Glu Asp Glu Glu Ala Gly Asn Gly Lys Asp 
            260                 265                 270 

Ser Ser Asn Asn Ser Lys Gly Gln Glu Gly Gln Asp His Gly Lys Glu 
        275                 280                 285 

Asp Asp His Asp Ser Ser Ile Gly Gln Asn Ser Asp Ser Lys Glu Tyr 
    290                 295                 300 

Tyr Asp Pro Glu Gly Lys Glu Asp Pro His Asn Glu Val Asp Gly Asp 
305                 310                 315                 320 

Lys Thr Ser Lys Ser Glu Glu Asn Ser Ala Gly Ile Pro Glu Asp Asn 
                325                 330                 335 

Gly Ser Gln Arg Ile Glu Asp Thr Gln Lys Leu Asn His Arg Glu Ser 
            340                 345                 350 

Lys Arg Val Glu Asn Arg Ile Thr Lys Glu Ser Glu Thr His Ala Val 
        355                 360                 365 

Gly Lys Ser Gln Asp Lys Gly Ile Glu Ile Lys Gly Pro Ser Ser Gly 
    370                 375                 380 

Asn Arg Asn Ile Thr Lys Glu Val Gly Lys Gly Asn Glu Gly Lys Glu 
385                 390                 395                 400 

Asp Lys Gly Gln His Gly Met Ile Leu Gly Lys Gly Asn Val Lys Thr 
                405                 410                 415 

Gln Gly Glu Val Val Asn Ile Glu Gly Pro Gly Gln Lys Ser Glu Pro 
            420                 425                 430 

Gly Asn Lys Val Gly His Ser Asn Thr Gly Ser Asp Ser Asn Ser Asp 
        435                 440                 445 

Gly Tyr Asp Ser Tyr Asp Phe Asp Asp Lys Ser Met Gln Gly Asp Asp 
    450                 455                 460 

Pro Asn Ser Ser Asp Glu Ser Asn Gly Asn Asp Asp Ala Asn Ser Glu 
465                 470                 475                 480 

Ser Asp Asn Asn Ser Ser Ser Arg Gly Asp Ala Ser Tyr Asn Ser Asp 
                485                 490                 495 

Glu Ser Lys Asp Asn Gly Asn Gly Ser Asp Ser Lys Gly Ala Glu Asp 
            500                 505                 510 

Asp Asp Ser Asp Ser Thr Ser Asp Thr Asn Asn Ser Asp Ser Asn Gly 
        515                 520                 525 

Asn Gly Asn Asn Gly Asn Asp Asp Asn Asp Lys Ser Asp Ser Gly Lys 
    530                 535                 540 

Gly Lys Ser Asp Ser Ser Asp Ser Asp Ser Ser Asp Ser Ser Asn Ser 
545                 550                 555                 560 

Ser Asp Ser Ser Asp Ser Ser Asp Ser Asp Ser Ser Asp Ser Asn Ser 
                565                 570                 575 

Ser Ser Asp Ser Asp Ser Ser Asp Ser Asp Ser Ser Asp Ser Ser Asp 
            580                 585                 590 

Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser 
        595                 600                 605 

Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Lys Ser 
    610                 615                 620 

Asp Ser Ser Lys Ser Glu Ser Asp Ser Ser Asp Ser Asp Ser Lys Ser 
625                 630                 635                 640 

Asp Ser Ser Asp Ser Asn Ser Ser Asp Ser Ser Asp Asn Ser Asp Ser 
                645                 650                 655 

Ser Asp Ser Ser Asn Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser 
            660                 665                 670 

Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Ser Ser Ser Asp 
        675                 680                 685 

Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser 
    690                 695                 700 

Ser Glu Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Asp Ser Ser Asp 
705                 710                 715                 720 

Ser Ser Asp Ser Ser Asn Ser Asn Ser Ser Asp Ser Asp Ser Ser Asn 
                725                 730                 735 

Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser 
            740                 745                 750 

Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser 
        755                 760                 765 

Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp 
    770                 775                 780 

Ser Asn Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser 
785                 790                 795                 800 

Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 
                805                 810                 815 

Asp Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser 
            820                 825                 830 

Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 
        835                 840                 845 

Asp Ser Ser Asp Ser Asp Ser Ser Asn Arg Ser Asp Ser Ser Asn Ser 
    850                 855                 860 

Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser 
865                 870                 875                 880 

Asp Ser Ser Asp Ser Ser Asp Ser Asn Glu Ser Ser Asn Ser Ser Asp 
                885                 890                 895 

Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Asp Ser Ser Asp Ser Ser 
            900                 905                 910 

Asn Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Glu 
        915                 920                 925 

Ser Ser Asn Ser Ser Asp Asn Ser Asn Ser Ser Asp Ser Ser Asn Ser 
    930                 935                 940 

Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser 
945                 950                 955                 960 

Asn Ser Gly Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Asn Ser 
                965                 970                 975 

Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser 
            980                 985                 990 

Asp Ser Ser Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asn  Ser Ser Asp 
        995                 1000                 1005 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asn  Ser Ser Asp 
    1010                 1015                 1020 

Ser Ser  Asn Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1025                 1030                 1035 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asn Ser Ser Asp  Ser Ser Asp 
    1040                 1045                 1050 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Gly  Ser Ser Asp 
    1055                 1060                 1065 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1070                 1075                 1080 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Glu  Ser Ser Asp 
    1085                 1090                 1095 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1100                 1105                 1110 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1115                 1120                 1125 

Ser Ser  Asn Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1130                 1135                 1140 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1145                 1150                 1155 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asp  Ser Ser Asp 
    1160                 1165                 1170 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Asn Glu  Ser Ser Asp 
    1175                 1180                 1185 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Ser Asn  Ser Ser Asp 
    1190                 1195                 1200 

Ser Ser  Asp Ser Ser Asp Ser  Ser Asp Ser Thr Ser  Asp Ser Asn 
    1205                 1210                 1215 

Asp Glu  Ser Asp Ser Gln Ser  Lys Ser Gly Asn Gly  Asn Asn Asn 
    1220                 1225                 1230 

Gly Ser  Asp Ser Asp Ser Asp  Ser Glu Gly Ser Asp  Ser Asn His 
    1235                 1240                 1245 

Ser Thr  Ser Asp Asp 
    1250 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            3 

tgcaaaagtc catgacagtg                                                 20 

 
           
             4  
             24  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            4 

tcagttggtt ctgagtaaaa agga                                            24 

 
           
             5  
             23  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            5 

aagtaatttt gtgctgttcc ttt                                             23 

 
           
             6  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            6 

aacaaagtga agaggttttc t                                               21 

 
           
             7  
             22  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            7 

aagaaccttt tcaattgcta gt                                              22 

 
           
             8  
             24  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            8 

tggagaagtt aatggaatgt agca                                            24 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            9 

tgcaatttgc tttccttcaa                                                 20 

 
           
             10  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            10 

cctcttcgtt tgctaatgtg g                                               21 

 
           
             11  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            11 

tcacaaggta gaagggaatg                                                 20 

 
           
             12  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            12 

gtttgtggct ccagcattgt                                                 20 

 
           
             13  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            13 

gggacacagg aaaagcagaa                                                 20 

 
           
             14  
             24  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            14 

tgttattgct tccagctact tgag                                            24 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            15 

caatgaggat gtcgctgttg                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            16 

tatccaggcc agcatcttct                                                 20 

 
           
             17  
             22  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            17 

cacctcagat caacagcaag ag                                              22 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            18 

tcttctttcc catggtcctg                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            19 

atgaagaagc agggaatgga                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            20 

attctttggc tgccattgtc                                                 20 

 
           
             21  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            21 

tgatggagac aagacctcca a                                               21 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            22 

tgccattgaa agaaatcagc                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            23 

ttctttcctc catccttcca                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            24 

ttctgatttt tggccaggtc                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            25 

ggcaatgtca agacacaagg                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            26 

tctcctcggc tactgctgtt                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            27 

tgcaaggaga tgatcccaat                                                 20 

 
           
             28  
             22  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            28 

tgtcatcatt cccattgtta cc                                              22 

 
           
             29  
             22  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            29 

caaaaggagc agaagatgat ga                                              22 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            30 

tgctgtcact gtcactgctg                                                 20 

 
           
             31  
             24  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            31 

gcagtgatag tagtgacagc agtg                                            24 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            32 

ttgctgctgt ctgacttgct                                                 20 

 
           
             33  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            33 

caaatcagac agtggcaaag g                                               21 

 
           
             34  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            34 

gctctcactg ctattgctgc t                                               21 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            35 

gcaagtcaga cagcagcaaa                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            36 

ctgctgtcgc tatcactgct                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            37 

atagcaacga cagcagcaat                                                 20 

 
           
             38  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            38 

tcgctgctat tgctatcact g                                               21 

 
           
             39  
             21  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            39 

gcaacagcag tgatagtgac a                                               21 

 
           
             40  
             18  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            40 

ctgctgtcgc tgctttca                                                   18 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            41 

agcagcgaca gcagtgatat                                                 20 

 
           
             42  
             22  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            42 

ttgttaccgt taccagactt gc                                              22 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            43 

tgacagcaca tctgacagca                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Primer  
             
           
            44 

tcccccagtt gtttttgttt                                                 20

Technology Classification (CPC): 6