Patent Publication Number: US-2009233272-A1

Title: Multiplexed Nucleic Acid Analysis with High Specificity

Description:
This application claims the benefit of our U.S. Provisional Patent Applications with the Ser. Nos. 60/532,681 (filed Dec. 23, 2003) and 60/556,737 (filed Mar. 26, 2004), both of which are incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     The field of the invention is genetic diagnostics, and especially as it relates to multiplex analysis of a single sample. 
     BACKGROUND OF THE INVENTION 
     Despite recent advances in molecular diagnostics, numerous difficulties still remain. Among other problems, analysis of multiple potential genetic changes in a sample suspected to include a virus or oncogene frequently lead to false positive results, or fail to identify all potential changes as the number of such changes increases. Similar difficulties arise where one or more organisms are subject to genotyping or other genetic analysis. 
     For example, human papillomavirus (HPV) is now considered a major cause of cervical cancer, killing more than 200,000 women around the world each year. The HPV virus is relatively common and more than 100 distinct types of HPV have been identified, some of which are considered “high-risk” for the development of cancer. Detection of such high-risk types of HPV has significant impact on diagnosis, prevention, treatment and management of cervical cancer in HPV-infected women. 
     To date, most molecular methods for HPV detection and typing rely on hybridization technologies, including southern blot, dot blot, line blot, and in situ hybridization. For example, HybridCapture II from Digene is a nucleic acid hybridization microplate assay based on chemiluminescence for the qualitative detection, and differentiating low-risk from high-risk groups. Other commercially available tests employ similar methods and may detect the presence of various types of HPV in a patient sample. However, known HPV typing methods based on hybridization often lack specificity due to cross-hybridization. Cross-hybridization may result in a false positive signal due to closely related types of HPV (e.g., where a target DNA has only a single or few mismatches to the probes being used). Thus, the accuracy of the test results may be compromised with samples containing multiple viral types with closely related sequences. 
     To overcome problems associated with cross-hybridization, a number of approaches have been taken. Typically, most of the improvements focus on exact control of the stringency conditions. For example, the specificity of hybridization can be controlled by temperature. However, temperature-specific hybridization may lead to false positive results if probes have a high degree of sequence similarity. 
     Other efforts included the use of peptide nucleic acids (PNA), a universal base stretch, or modified bases (e.g., super G and C) to alter or otherwise affect hybridization/melting temperature of duplexes. Still further known methods involve use of conformationally locked DNA (e.g., to increase duplex stability), etc. While most of such approaches have provided at least some advantages, various problems nevertheless remain. Among other things, currently known approaches tend to fail to provide a significant difference between the melting and/or hybridization temperature of a perfectly matched hybrid and a single base mismatched hybrid. 
     Therefore, while numerous methods for nucleic acid based testing of HPV and other pathogens are known in the art, all or almost all of them suffer from various problems, which are even more aggravated, where such analysis is performed in a multiplex environment (e.g., a biochip). Consequently, there is still a need to provide improved methods and compositions for molecular diagnostics. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to compositions and methods for genetic diagnostics in which specificity is substantially improved by using a combination of selected multiplex amplification primers and selected multiplex extension primers, wherein the sequences of the primers are designed to maximize hybridization specificity and extension selectivity in a multiplex reaction. 
     In one aspect of the inventive subject matter, a multiplex diagnostic kit includes a plurality of amplification primer pairs, and a plurality of extension primers, wherein each of the plurality of amplification primer pairs has a sequence such that (a) a plurality of amplicons produced from a target nucleic acid using the plurality of amplification primer pairs, respectively, includes a sequence difference (mutated position) in a target nucleic acid, (b) the plurality of amplicons is produced in a PCR reaction using the same amplification profile, and wherein each of the plurality of extension primers has a sequence such that (c) each of the plurality of extension primers specifically hybridizes to each of the plurality of amplicons at the same extension temperature, respectively, and selective primer extension for each of the plurality of extension primers is achieved at the same extension profile. Amplification primers and extension primers are most preferably those described in SEQ ID Ax, SEQ ID Bx, and SEQ ID Cx, with x being an integer between 1 and 24. 
     Particularly preferred kits further include a biochip to which are coupled in a plurality of distinct positions a plurality of distinct capture probes, respectively, and wherein each of the plurality of capture probes hybridizes with a portion of each of the extension primers, respectively. Most preferably, each of the plurality of the distinct capture probes has a unique sequence distinct from the target nucleic acid. Additionally, contemplated kits can include DNA-dependent DNA polymerase (e.g. thermostable, or specifically modified and/or isolated for primer extension), and/or an instruction (e.g., to perform the PCR reaction and primer extension in a single tube). 
     In another aspect of the inventive subject matter, a multiplex diagnostic kit includes at least two forward amplification primers having a sequence according to SEQ ID Ax and Ay, at least two backward amplification primers having a sequence according to SEQ ID Bx and By, and at least two extension primers having a sequence according to SEQ ID Cx and Cy, wherein x and y are integers between 1 and 24 and not the same. Such kits can further include an instruction to perform a multiplex PCR using the at least two forward amplification primers and the at least two backward amplification primers using the same amplification profile, and optionally an instruction to perform a primer extension reaction using the at least two extension primers at the same extension profile (typically in a single test tube). 
     Additionally, or alternatively, a biochip is included in the test kit to which are coupled in a plurality of distinct positions a plurality of distinct capture probes, respectively, and wherein each of the plurality of capture probes hybridizes with a portion of each of the extension primers, respectively. Most preferably, each of the plurality of the distinct capture probes has a unique sequence distinct from a target nucleic acid to which the amplification primers bind. Where desired, contemplated kits may also include a reagent and/or an enzyme. 
     In a still further contemplated aspect of the inventive subject matter, a synthetic nucleic acid has less than sixty (most preferably less than forty) nucleotides and comprises an HPV recognition sequence selected from the group consisting of SEQ ID Ax, SEQ ID Bx, and SEQ ID Cx, wherein X is an integer between 1 and 24, wherein no more than two nucleotides in the HPV recognition sequence are replaced by N (A, G, C, or T). Such synthetic nucleic acids especially include those having SEQ ID Cx and further comprise a plurality of nucleotides at the 5′-terminus that have less than 60% homology to a target sequence to which the nucleic acid hybridizes. 
     Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention. 
    
    
     DETAILED DESCRIPTION 
     The inventors have unexpectedly discovered that a plurality of potential variants of a single gene can be identified in a single sample using a multiplex test in which amplification primers are used to specifically amplify a target sequence in that gene, wherein the amplicon includes at least one of the potential variants, and wherein extension primers are used to form an extension product that is specific to a variant of the gene. 
     It should be especially noted that the specificity in such tests is substantially increased over conventional methods by the manner of primer selection. Specifically, the amplification primers are selected to have a sequence such that (a) a plurality of amplicons produced from a target nucleic acid using the amplification primers include sequence difference in a target nucleic acid, and (b) the plurality of amplicons is produced in a PCR reaction using the same amplification profile. In the same test, the extension primers are selected to have a sequence such that (c) the extension primers specifically hybridize to the corresponding amplicons at the same extension temperature (preferably such that the 3′-end of each of the extension primers corresponds to a complementary position of the mutated position), and (d) selective primer extension for each of the extension primers is achieved at the same extension temperature. 
     Most preferably, the 5′-end of the extension primers further includes a tag (zipcode sequence) that is substantially not (typically less than 70%, and most typically less than 50%) complementary to the sequence of the amplicons and/or the sequence of the target gene, wherein the zipcode sequence is employed to hybridize with a capture probe (preferably on a biochip in a predetermined position). While not limiting to the inventive subject matter, the zipcode has typically a length between about two and twenty, more preferably between five and fifteen, and most preferably between eight and twelve nucleotides, wherein the tags of each of the extension primers are distinct (i.e., have a unique sequence), and wherein the zipcodes (and with that the distinct capture probes) have a unique sequence distinct from the target nucleic acid. 
     With respect to the particular sequences of the amplification primers and the extension primers, it should be recognized that all sequences are deemed suitable and that the specific sequences will predominantly depend on the particular nature of the target nucleic acid and type of sequence difference that is to be detected. For example, suitable target nucleic acids include native and recombinant DNA (e.g., linear, circular, etc.), RNA (e.g., snRNA, hnRNA, niRNA, etc.), synthetic nucleic acids (e.g., phosphorothioates, PNA, etc.), all of which may be present in, or isolated from a biological source (e.g., biopsy, cell culture, swab, filtrate, plant material, etc.), a non-biological source (e.g., food, soil, water, oil, etc.), or may be entirely synthetic (e.g., on solid phase). Thus, it should be recognized that the length of contemplated target nucleic acids may vary considerably, and is typically between about 50 nucleotides to the length of an entire genome, chromosome, vector, chromosomal fragment, or transcript. Most preferably, the target nucleic acid is a viral or bacterial genome, or a nucleic acid comprising an oncogene, tumor suppressor gene, or other gene that is associated with a predisposition or presence of a disease. In the example, below, a particularly preferred target DNA is a viral DNA, and especially HPV DNA. 
     It is generally preferred that the amplification primers have a length of between about 12 to 50 nucleotides, and more preferably between about 16 to 30 nucleotides, wherein the amplification primers may additionally (or optionally) include one or more nucleotides that provide one or more desirable properties. For example, contemplated amplification primers may include one or more nucleotides that render the primer (and/or amplicon) quantifiable and typical examples include radiolabeled nucleotides, fluorescence-labeled nucleotides, etc. In another example, contemplated amplification primers may also include one or more nucleotides that will facilitate specific isolation of the primer and/or amplicon (e.g., biotinylated nucleotide). Thus, amplicons generated by contemplated methods may be quantified to normalize a test result, especially where the test result provides a quantitative measure. Amplicons generated by contemplated tests will typically have a length of between about 50 to several thousand nucleotides. 
     Similarly, preferred extension primers can have a length of between about 12 to 50 nucleotides, and more preferably between about 16 to 30 nucleotides, wherein the extension primers may additionally (or optionally) include one or more nucleotides that provide for one or more desirable properties. For example, particularly contemplated extension primers can include several additional nucleotides that allow specific hybridization of the additional nucleotides to a capture probe. Most preferably, the additional nucleotides have a sequence that is distinct from the sequence of the target nucleic acid (and even more typically of the amplicon). Therefore, capture of the extension product is independent of the target sequence, which further increases selectivity of the test. For example, various SNP-specific tests known in the art use solid-phase or otherwise immobilized extension primers, which tend to produce false positive results where the sequence difference among various mutant sequences allows cross-hybridization. Of course, it should be recognized that the sequence of the extension primer is selected from a sequence available in the amplicon. 
     Depending on the particular target nucleic acid, contemplated amplification and/or extension primers can also include modified nucleotides and/or have one or more ambiguous positions (i.e., a position in which different nucleotides are present among otherwise identical primers). Ambiguous positions are denoted using the IUPAC nomenclature (R is A or G, Y is C or T, S is C or G, W is A or T, K is G or T, M is A or C, B is C or G or T, D is A or G or T, H is A or C or T, V is A or C or G, and N is A or C or G or T). Therefore, contemplated amplification and/or extension primers may have a single defined T m  at a particular solvent and temperature, or several distinct T m . 
     However, it should be recognized that the amplification primers are chosen such that a multiplex PCR using the amplification primers can be performed using a single amplification profile (wherein the term “amplification profile” refers to a specific combination of denature temperature and time, anneal temperature and time, and polymerization temperature and time), and that all amplicons produced from the multiplex PCR can be used for a primer extension reaction using the extension primers at a single extension temperature (wherein the term “extension temperature” refers to a specific combination of hybridization temperature and time and polymerization temperature and time). Preferred extension products are typically in the range of about 50 to several thousand bases, and it is especially preferred that the extension product includes one or more detectable (and more preferably quantifiable) label. For example, the extension reaction may be performed using one or more directly or indirectly labeled nucleotides, including nucleotides that carry a fluorescent, luminescent, or radioactive label (wherein the molar fraction of labeled nucleotide may be adjusted as appropriate), and/or nucleotides that carry an affinity marker (e.g., biotin, digitoxin) that binds a labeled compound or compound that can otherwise be detected and/or quantified. 
     It is generally preferred that the extension primer has a sequence and is positioned such that proper hybridization of the extension primer (and especially correct hybridization of the terminal three 3′ bases, more preferably terminal two 3′ bases, and most preferably terminal 3′ base) with the target nucleic acid will result in a detectable extension event. Typically the detectable event is a DNA polymerase-dependent DNA synthesis, wherein at least one of the nucleotides is labeled. Alternatively, the detectable event may also be a DNA ligation using a labeled fragment that abuts with it&#39;s 5′-end the 3′-end of the extension primer. With respect to the type of sequence difference that can be detected using contemplated methods, it should be recognized that all known sequence differences are suitable so long as information is available that allows design of the amplification primers and the extension primers. Thus, contemplated differences include deletions, insertions, translocations, and substitutions (e.g., transversion or transition). Furthermore, it should be noted that contemplated sequence differences also include sequence differences found in distinct viral genotypes. Thus, the nucleotide differences between or among various genotypes of a viral species are also considered mutations herein. 
     Detection is preferably carried out on a biochip or other carrier onto which are immobilized in predetermined positions a plurality of capture probes that hybridize with at least a portion of the extension primer and/or extension product. Therefore, contemplated diagnostic kits can also include (next to contemplated amplification primers and/or extension primers) a biochip to which are coupled in a plurality of distinct positions a plurality of distinct capture probes, respectively, and wherein each of the plurality of capture probes hybridizes with at least a portion of each of the extension primers, respectively. In still further preferred aspects, contemplated capture probes may also include a fluorescent label, wherein the emission of the label is most preferably at a wavelength different from the detection wavelength of the extension product (e.g., Cy5 for the capture probe and Cy3 for the extension product). Among other advantages, such configurations allow normalization and/or calibration of a signal from the extension product. Additionally, or alternatively, suitable kits may include various enzymes (e.g., DNA-dependent DNA polymerase, ligase, etc.), buffers, and other reagents (e.g., labeled and unlabeled nucleotides). 
     Table 1A depicts exemplary forward and backward amplification primers and corresponding extension primers for detection of genetic variants of HPV, wherein an extension primer in the same row as a forward and backward amplification primer will bind to the amplicon produced by the amplification primers. It should be noted that the primers in the Table 1A may include degenerate nucleotide positions. Therefore, primers with degenerate positions represent both individual sequences as well as mixtures of sequences defined by the ambiguity codes (e.g., ASA may represent AGA individually or ACA individually, but also a mixture of ACA and AGA together). Table 1B depicts an exemplary selection of certain primers of Table 1A with non-degenerate sequences. 
     The amplification primers and extension primers correspond to the sequences provided in the sequence listing below, wherein SEQ ID: A1-A24 of Table 1A correspond to Sequence Numbers 1-24 of the sequence listing, respectively, wherein SEQ ID: B 1-B24 of Table 1 correspond to Sequence Numbers 25-48 of the sequence listing, respectively, and wherein SEQ ID: C1-C24 of Table 1 correspond to Sequence Numbers 49-72 of the sequence listing, respectively. Furthermore, SEQ ID: A25-A26 of Table 1B correspond to Sequence Numbers 73-74 of the sequence listing, respectively, and SEQ ID: B25-B41 of Table 1B correspond to Sequence Numbers 75-91 of the sequence listing, respectively. 
     The primers according to Tables 1A and 1B (and other primers contemplated above) can further be modified to yield a synthetic nucleic acid having less than seventy, more preferably less than sixty nucleotides, and most preferably less than 50 nucleotides and comprising an HPV recognition sequence selected from the group consisting of SEQ ID Ax, SEQ ID Bx, and SEQ ID Cx, wherein X is an integer between 1 and 24, wherein no more than three, and most preferably no more than two nucleotides in the HPV recognition sequence are replaced by N (as defined in IUPAC nomenclature) or other non-natural nucleotide. Furthermore, nucleotides according to SEQ ID Cx may further include a plurality of nucleotides at the 5′-terminus that have less than 70%, more typically less than 60%, and most typically less than 40% homology to a target sequence to which the nucleic acid hybridizes. 
     Especially contemplated kits will include at least two, more typically at least three to five, and most typically at least ten to twenty of the amplification primer pairs, and/or corresponding extension primers. In such kits, the PCR reaction and/or the primer extension is preferably performed in a single tube, which may be reflected in an instruction accompanying such kits. Alternatively, at least one of the PCR reaction and the primer extension can also be performed in an automated analyzer. Contemplated instructions may further provide information to perform the multiplex PCR using at least two forward amplification primers and at least two backward amplification primers using the same amplification profile, and/or information to perform the primer extension reaction using at least two extension primers at the same extension temperature. 
     While not wishing to be bound by a particular hypothesis or theory, the inventors contemplate that the specificity of the tests according to the inventive subject matter is further improved by virtue of the fact that at least one of the hybridizations, and more preferably both hybridizations (i.e., for amplification and extension) is performed in solution rather than on a solid phase (which is thought to interfere with hybridization specificity). Furthermore, by the particular choice of primer selection, and especially by targeting distinguishing sequences among a plurality of otherwise similar or identical sequences, hybridization specificity of the amplification and/or extension primers is further increased. 
     Experiments 
     The following experiments were performed to provide exemplary guidance for a test to detect and genotype an HPV virus from a human sample. Here, HPV DNA was isolated from a pap smear using the Qiagen DNA isolation kit and an aliquot of the eluent was subjected to an off-line multiplex PCR using the forward and backward amplification primers with the SEQ ID A1-A24 and B1-B24, respectively. The PCR conditions were as follows: 
     HPV Multiplex PCR 
     To 1 uL sample were added 18.75 uL HPV Amplification Solution, 0.25 uL (1.25 units) Platinum Taq Polymerase (Invitrogen) to a final volume of 20 uL. The HPV Amplification Solution was 21.34 mM Tris-HCL (pH 8.4), 53.35 mM KCL, 2.67 mM MgCl2, 33.34 uM dATP, 33.34 uM dGTP, 33.34 uM dTTP, 6.67 uM dCTP, and 26.68 to 80.03 nM for each of the forward and backward amplification primers. 
     A 24-plex PCR using the primers of Table 1A was performed using the following amplification profile: Activation of Platinum Taq polymerase was performed by incubation at 94° C. for 1 min followed by 40 cycles of 5 sec denaturation at 94° C., 30 sec annealing at 52° C. and 40 sec elongation at 72° C. The contents of the multiplex PCR was then used in a subsequent primer extension using the extension oligos with the SEQ ID C1-C24 in a single well of a multi-well plate that was disposed in an automated analyzer as follows: 
     HPV Primer Extension 
     The primer extension was performed in a 24 well plate in an automated analyzer using temperature controlled incubation of the extension mixture and reagents as follows: 
     To the 20 uL volume from the multiplex PCR reaction were added 20 uL HPV Primer Extension Solution to a final volume of 40 uL. The HPV Primer Extension Solution was 20 mM Tris-HCL (pH 8.4), 50 mM KCL, 2.5 mM MgCl2, 31.25 uM dATP, 31.25 uM dGTP, 31.25 uM dTTP, 5 uM cy5dCTP, and 25 nM for each of the extension primers SEQ ID C1-C24. The extension profile was as follows: The PCR reaction was denatured at 94° C. for 1 min followed by 40 cycles of 5 sec at 94° C. and 10 sec at 51° C. 
     HPV Detection and Genotyping 
     The entire volume of the extension reaction was then transferred onto a biochip that included in predetermined position a plurality of capture nucleotides as described in our copending International applications WO 03/050591 and WO 02/057416, both of which are incorporated by reference herein. Extension products were detected by fluorescence detection using the Cy label on the extension product (which is only formed where the extension primer forms a perfect hybrid with the target nucleic acid), wherein a predetermined position of the capture primer corresponds to a predetermined HPV genotype. Genotyping and detection was confirmed using the reference test HC2 HPV DNA test from Digene Corporation. The results from the test according to the inventive subject matter and the commercially available test correlated 100% as shown in Table 2 in which HR represents “high-risk” genotype (with particular genotype provided in parentheses), LR represents “low-risk” genotype (with particular genotype provided in parentheses), and neg represents negative result. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 INVENTIVE TEST (24 PLEX) 
                 REFERENCE TEST 
               
               
                   
                   
               
             
            
               
                   
                 HR (45) 
                 HR 
               
               
                   
                 neg 
                 neg 
               
               
                   
                 neg 
                 neg 
               
               
                   
                 HR (31, 66) 
                 HR, LR 
               
               
                   
                 HR (16, 26, 33, 52, 82), LR (6) 
                 HR, LR 
               
               
                   
                 HR (16) 
                 HR, LR 
               
               
                   
                 HR (35, 58, 66) 
                 HR, LR 
               
               
                   
                 HR (18, 53, 56), LR (42) 
                 HR, LR 
               
               
                   
                 HR (39, 58) 
                 HR 
               
               
                   
                 HR (35) 
                 HR 
               
               
                   
                 neg 
                 neg 
               
               
                   
                 HR (35, 52) 
                 HR 
               
               
                   
                 HR (18) 
                 HR 
               
               
                   
                 HR (18, 35) 
                 HR, LR 
               
               
                   
                   
               
            
           
         
       
     
     Thus, specific embodiments and applications of multiplexed HPV nucleic acid analysis with improved specificity have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 1A 
               
               
                   
               
               
                   
                 SEQ 
                   
                 SEQ 
                   
                 SEQ 
                   
                   
               
               
                 HPV 
                 ID. 
                 Upstream Primer 
                 ID. 
                 Downstream Primer 
                 ID. 
                 Extension Primer 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 6 
                 A1 
                 GCAACAACAGTTGAAGAAGAAAC 
                 B1 
                 AGCTGTTGCACTTCTCTGATGTC 
                 C1 
                 GAAGTGGACGGACAAGATTC 
                   
               
               
                   
               
               
                 11 
                 A2 
                 GCACCTACAGTAGAAGAAGAAAC 
                 B2 
                 AGGTCTTGTAGTTGTCTGATGTC 
                 C2 
                 CAAGGTGGACAACACAGACG 
               
               
                   
               
               
                 16 
                 A3 
                 GTATATAGAGAKGGRAATGC 
                 B3 
                 AATTGCTCATAACAGTRGAGRTCA 
                 C3 
                 GCATGGAGATACACCTACATTG 
               
               
                   
               
               
                 18 
                 A4 
                 GTGTATAGAGACAGTATACCG 
                 B4 
                 AATTGCTGGTGACATASAAGGTCA 
                 C4 
                 GACAGGAACGACTCCAACGAC 
               
               
                   
               
               
                 26 
                 A5 
                 GTATATAGAGATAGGAGTCC 
                 B5 
                 AATGTTCGTARCASYGTAGGTCA 
                 C5 
                 GAGACCAAGGCGCCAAACAG 
               
               
                   
               
               
                 31 
                 A6 
                 GTATATAGGGACGACACACC 
                 B6 
                 AATTGCTCATAACAGTRGAGRTCA 
                 C6 
                 CATGCGTGGAGAAACACCTACG 
               
               
                   
               
               
                 33 
                 A7 
                 GTATATAGAGAKGGRAATCC 
                 B7 
                 AATTGCTCATARCAGTATAGGTCA 
                 C7 
                 CGACGTAGAGAAACTGCACTG 
               
               
                   
               
               
                 35 
                 A8 
                 GTATATAGAGAAGGCCAGCC 
                 B8 
                 AATTGCTCATARCAGTATAGGTCA 
                 C8 
                 GACAGGTCGGTGTATGTCCT 
               
               
                   
               
               
                 39 
                 A9 
                 GTATATAGGGACGGGGAACC 
                 B9 
                 AATTGCTCGTGACATASAAGGTCA 
                 C9 
                 GACCGCAGACTAACACGAAGA 
               
               
                   
               
               
                 42 
                 A10 
                 TGGTATACAGTGGAGAAAGAAAC 
                 B10 
                 CCCAAAAGCATCTGTTGCAG 
                 C10 
                 TGACCAAGCCAAACAGGACA 
               
               
                   
               
               
                 43 
                 A11 
                 GCAGATACTGTAGAAGAAGAAAC 
                 B11 
                 CAGTCTTCTAGCTTCTTGATGTC 
                 C11 
                 GGACCAGCAAGTGAATCTAC 
               
               
                   
               
               
                 44 
                 A12 
                 GCAGTAACAGTGGAAGAAGAAAC 
                 B12 
                 AGCGTATGTAGGTGATGGATGTC 
                 C12 
                 CGCAAGACGTTACACAGCCT 
               
               
                   
               
               
                 45 
                 A13 
                 GTGTATAGAGACTGTATAGC 
                 B13 
                 AATGCTCGTARCACAMMAGGTCA 
                 C13 
                 GAAAGACTTCGCAGACGTAGG 
               
               
                   
               
               
                 51 
                 A14 
                 GATTGTATATAGGGATAATAATCCAT 
                 B14 
                 AATTGCTCRTRGCATTGCARGTCA 
                 C14 
                 ACGTACACGACAACGTAACG 
               
               
                   
               
               
                 52 
                 A15 
                 GAATAGTATATAGAGACAATAATCC 
                 B15 
                 AATTGCTCATAGCAGWRTAGGTCA 
                 C15 
                 CTGTGACCCAAGTGTAACGTC 
               
               
                   
               
               
                 53 
                 A16 
                 GTGTATAGAGACGGGTATCC 
                 B16 
                 AATTGCTCRTRGCATTGCARGTCA 
                 C16 
                 GACCGGGTCGTGCCTGAC 
               
               
                   
               
               
                 56 
                 A17 
                 GTGTATAGGGATGATTTTCC 
                 B17 
                 AATTGCTCATTGCAYTGTAGGTCA 
                 C17 
                 GAGACAAACATCTAGAGAACC 
               
               
                   
               
               
                 58 
                 A18 
                 GTGTATAGAGATGGAAATCC 
                 B18 
                 AATTGCTCATAGCAGWRTAGGTCA 
                 C18 
                 GACAGGGCGCTGTGCAGTG 
               
               
                   
               
               
                 59 
                 A19 
                 GTGTATAGAGACTGTAGACC 
                 B19 
                 AATTGCTCGTARCACAMMAGGTCA 
                 C19 
                 CAAAGACAAGCGCGTAGTG 
               
               
                   
               
               
                 66 
                 A20 
                 AACTAGTATATAGAAACATTGGC 
                 B20 
                 AATTGCTCATTGCAYTGTAGGTCA 
                 C20 
                 GACATACGAGTAGACAAGCTACAG 
               
               
                   
               
               
                 68 
                 A21 
                 GTAGTATATAGGGACGGGGTA 
                 B21 
                 AATTGCTCGTGACATACAAGGTCG 
                 C21 
                 GCAGACGCACACGGCAGG 
               
               
                   
               
               
                 69 
                 A22 
                 GAATAGTGTATAGAAATGATAGTGC 
                 B22 
                 AATTGTTCGTARCASYGTAGGTCA 
                 C22 
                 GGATGAAAAGCGACGGTTCC 
               
               
                   
               
               
                 73 
                 A23 
                 GTATTGTATATAGAAAGGATAAACC 
                 B23 
                 CAATGACTCGTAACATGTAAGGTC 
                 C23 
                 CGGTTTCATCAAATAGCAGAACA 
               
               
                   
               
               
                 82 
                 A24 
                 GTATATAGGACAATACGCC 
                 B24 
                 AATTGCTCRTRGCATTGCARGTCA 
                 C24 
                 GTGAAACCCAGGTGTAATAACG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 1B 
               
               
                   
               
               
                   
                 SEQ 
                   
                 SEQ 
                   
                 SEQ 
                   
                   
               
               
                 HPV 
                 ID. 
                 Upstream Primer 
                 ID. 
                 Downstream Primer 
                 ID. 
                 Extension Primer 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 16 
                 A25 
                 GTATATAGAGATGGGAATCC 
                 B25 
                 AATTGCTCATAACAGTAGAGATGA 
                 G3 
                 GCATGGAGATACACCTACATTG 
                   
               
               
                   
               
               
                 18 
                 A4 
                 GTGTATAGAGACAGTATACCG 
                 B26 
                 AATTGCTCGTGACATAGAAGGTCA 
                 C4 
                 GACAGGAACGACTCCAACGAC 
               
               
                   
               
               
                 26 
                 A5 
                 GTATATAGAGATAGGAGTCC 
                 B27 
                 AATTGTTCGTAGCAGCGTAGGTCA 
                 C5 
                 GAGACCAAGGCGCCAAACAG 
               
               
                   
               
               
                 31 
                 A6 
                 GTATATAGGGACGACACACC 
                 B28 
                 AATTGCTCATAACAGTGGAGGTCA 
                 C6 
                 CATGCGTGGAGAAACACCTACG 
               
               
                   
               
               
                 33 
                 A26 
                 GTATATAGAGAGGGAAATCC 
                 B29 
                 AATTGCTCATAGCAGTATAGGTCA 
                 C7 
                 CGACGTAGAGAAACTGCACTG 
               
               
                   
               
               
                 35 
                 A8 
                 GTATATAGAGAAGGCCAGCC 
                 B30 
                 AATTGCTCATAACAGTATAGGTCA 
                 C8 
                 GACAGGTCGGTGTATGTCCT 
               
               
                   
               
               
                 39 
                 A9 
                 GTATATAGGGACGGGGAACC 
                 B31 
                 AATTGCTCGTGACATACAAGGTCA 
                 C9 
                 GACCGCAGACTAACACGAAGA 
               
               
                   
               
               
                 45 
                 A13 
                 GTGTATAGAGACTGTATAGC 
                 B32 
                 AATTGCTCGTAACACAACAGGTCA 
                 C13 
                 GAAAGACTTCGCAGACGTAGG 
               
               
                   
               
               
                 51 
                 A14 
                 GATTGTATATAGGGATAATAATCCAT 
                 B33 
                 AATTGCTCGTAGCATTGCAAGTCA 
                 C14 
                 ACGTACACGACAACGTAACG 
               
               
                   
               
               
                 52 
                 A15 
                 GAATAGTATATAGAGACAATAATCC 
                 B34 
                 AATTGCTCATAGCAGTGTAGGTCA 
                 C15 
                 CTGTGACCCAAGTGTAACGTC 
               
               
                   
               
               
                 53 
                 A16 
                 GTGTATAGAGACGGGTATCC 
                 B35 
                 AATTGCTCATGGCATTGCAGGTCA 
                 C16 
                 GACCGGGTCGTGCGTGAC 
               
               
                   
               
               
                 56 
                 A17 
                 GTGTATAGGGATGATTTTCC 
                 B36 
                 AATTGCTCATTGCACTGTAGGTCA 
                 C17 
                 GAGACAAACATCTAGAGAACC 
               
               
                   
               
               
                 58 
                 A18 
                 GTGTATAGAGATGGAAATCC 
                 B37 
                 AATTGCTCATAGCAGAATAGGTCA 
                 C18 
                 GACAGGGCGCTGTGCAGTG 
               
               
                   
               
               
                 59 
                 A19 
                 GTGTATAGAGACTGTAGACC 
                 B38 
                 AATGCTCGTAGCACACAAGGTCA 
                 C19 
                 CAAAGACAAGCGCGTAGTG 
               
               
                   
               
               
                 66 
                 A20 
                 AACTAGTATATAGAAACAATTGGC 
                 B39 
                 AATTGCTCATTGCATTGTAGGTCA 
                 C20 
                 GACATACGAGTAGACAAGCTACAG 
               
               
                   
               
               
                 69 
                 A22 
                 GAATAGTGTATAGAAATGATAGTGC 
                 B40 
                 AATTGTTCGTAACACTGTAGGTCA 
                 C22 
                 GGATGAAAAGCGACGGTTCC 
               
               
                   
               
               
                 82 
                 A24 
                 GTATATAGGACAATACGCC 
                 B41 
                 AATTGCTCGTAGCATTGCAAGTCA 
                 C24 
                 GTGAAACCCAGGTGTAATAACG