Detection of human papillomavirus by the polymerase chain reaction

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides medical research and diagnostic methods for detecting and typing HPV.
The methods utilize PCR, a DNA amplification technique widely used in the fields of molecular biology and genetic engineering. The methods can also be used to generate information concerning previously unknown types and sub-types of HPV and consequently has applications in the field of virology. 2. Description of Related Art
Papillomaviruses have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa. And although genital HPV infection is associated with cancer primarily in women, recent evidence suggests that HPV may play a role in the development of anogenital cancers in men. Broker et al., 1986, Cancer Cells 4:17-36, review the molecular, cellular, and clinical aspects of the papillomaviruses and the relationship of HPVs to cancer. HPV types 6, 11, 16, 18, and 33 are known genital HPV types in the human population, and Broker et al., 1986, Cancer Cells 4:589-594, disclose that HPV types 6, 11, 16, 18, and 33 share significant homology at the DNA level, particularly at the L1 open reading frame.
Identification and typing of HPV is quite important, because different types of HPV pose different risks to the affected individuals. For instance, HPV 16 and HPV 18 have been more consistently identified in higher grades of cervical dysplasia and carcinoma than other HPV types. Webb et al., December 1987, J. Inf. Disease 156(6):912-919, report a method for detecting HPV DNA types that utilizes a reverse-blotting procedure. The procedure involved forming a membrane to which genomic DNA from four different HPV types was bound and then hybridizing labelled DNA from a biological sample to the DNA bound to the membrane. Caussey et al., February 1988, J. Clin. Microbiol. 26(2):236-243 describe similar HPV detection methods.
Shibata et al., January 1988, J. Exp. Med. 167:225-230, disclose the use of PCR to amplify and detect the presence of HPV 16 and HPV 18 DNA. U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose PCR and the use of PCR to detect the presence or absence of nucleic acid sequence in a sample. European Patent Publication Nos. 229,701 and 269,445 disclose the use of PCR to amplify and detect DNA sequences associated with a wide variety of viruses, including the AIDS virus, HTLV I, and HTLV II.
Maitland et al., May 1988, Seventh International Papillomavirus Workshop, Abstract, p. 5, report the use of PCR to detect HPV16 in oral and cervical biopsies. In addition, Campione-Piccardo et all., May 1988, Seventh International Papillomavirus Workshop, Abstract, p. 19, report the use of a mixture of primers for the specific amplification by PCR of HPV sequences in types 1a, 5, 6a, 6b, 8, 11, 16, 18, and 33. A number of other researchers disclosed the use of PCR to amplify and detect HPV sequences at the Seventh International Papillomavirus Workshop. Each of the background references described in this section is incorporated herein by reference.
The heterogeneity of the human papillomavirus group is generally described in deVilliers, 1989, J. Virology 63:4898-4903, which is incorporated herein by reference. The genomic of numerous HPV types have been sequenced and/or characterized. For example, for HPV type 6, see deVilliers et al., 1981, J. Virology 40:932-935, and Gissmann and Zur Hausen, 1980, Int. J. Cancer 25:605-609. For HPV type 2, see Gissmann et al., 1982, J. Virology 44:393-400. For HPV type 16, see Seedoff et al., 1985, Virology 145:181-185. For HPV type 18, see Cole and Danos, 1987, J. Mol. Biol. 193:599-608. For HPV type 31, see Goldsborough et al., 1989, Virology 171:306-311. For HPV 33, see Cole and Streeck, 1986, J. Virology 58:991-995. For HPV 54, see Favre et al., 1990, Int. J. Cancer 45:40-46. For HPV 56, see Lorinez, 1989, J. Gen. Virol. 70:3099. These publications are incorporated herein by reference.
Despite the use of PCR to amplify and detect HPV sequences, there still remains a need for a simple and rapid method for both detecting and typing HPV in a biological sample. The present invention provides a method that meets that need.
SUMMARY OF THE INVENTION
The present invention provides methods for detecting and typing HPV in a sample. The methods comprise amplifying a sequence of HPV DNA present in the sample, determining if amplification has occurred, and then hybridizing an HPV type-specific probe to the amplified DNA. The invention also provides novel primers and probes for use in the method. In a preferred embodiment long, generic probes are used for determining if amplification has occurred.

DETAILED DESCRIPTION
The present invention provides a method for detecting HPV in a sample, and typing the HPV if present, the method comprising:
(a) treating said sample with consensus HPV primers, an agent for polymerization, and deoxynucleoside 5'-triphosphates under hybridizing conditions, wherein said consensus HPV primers are mixtures of oligonucleotides that comprises at least a pair of primers sufficiently complementary to separate strands of the nucleic acid genome for each member of the group of HPV types that includes HPV-6, - 11, - 16, -18, and -33 to hybridize therewith, such that the extension product synthesized from one member of said pair, when separated from its complementary strand, can serve as a template for synthesis of the extension product of the other member of said pair;
(b) treating the sample under denaturing conditions to separate the primer extension products from their templates, providing single-stranded molecules;
(c) treating the products of step (b) with the consensus primers of step (a) under conditions such that a primer extension product is synthesized using each of the single-stranded molecules produced in step (b) as a template;
(d) repeating steps (b) and (c) to synthesize detectable amounts of said HPV nucleic acid, if present in said sample, providing an amplified HPV nucleic acid sequence;
(e) determining if amplification has occurred by treating the reaction mixture prepared in step (d) under hybridizing conditions with a long generic probe and determining if hybridization has occurred; and, if amplification has occurred,
(f) hybridizing a type-specific DNA probe to said amplified DNA and determining if hybridization has occurred.
The first step in the method of the invention requires the use of consensus primer pairs that will amplify a discrete region of DNA from HPV DNA present in a sample. These consensus primer pairs are oligonucleotides, and the consensus primers may be mixtures of primer pairs. The mixtures used in the method assure that, regardless of the type of HPV present in a sample, HPV DNA corresponding to the region between the "consensus" sequences will be amplified. The PCR products generated from the consensus primers, if HPV is present in the sample, are then analyzed by hybridization with type-specific probes to determine the HPV types present.
Amplification of DNA by the polymerase chain reaction (PCR) is disclosed in U.S. Pat. Nos. 4,683,202 and 4,683,195 and incorporated herein by reference. PCR amplification of DNA involves repeated cycles of heat-denaturing the DNA, annealing two oligonucleotide primers to sequences that flank the DNA segment to be amplified, and extending the annealed primers with DNA polymerase. The primers hybridize to opposite strands of the target sequence and are oriented so DNA synthesis by the polymerase proceeds across the region between the primers, effectively doubling the amount of that DNA segment. Moreover, because the extension products are also complementary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. This results in the exponential accumulation of the specific target fragment, at a rate of approximately 2.sup.n per cycle, where n is the number of cycles.
The choice of primers for use in PCR determines the specificity of the amplification reaction. In the amplification steps of the method of the present invention, "consensus" primers are used that will amplify HPV sequences present in a sample, regardless of type. The consensus primers of the invention can include degenerate primers, mixtures of the oligonucleotides synthesized so that any one of several nucleotides can be incorporated into a primer at a selected position during synthesis. The consensus primers are sufficiently complementary to all types of HPVs to amplify a DNA sequence of any HPV present in the sample. The consensus primers are also designed to amplify a region of DNA that contains sequences that are specific to each major viral type, so the amplified DNA can therefore be used to type the HPV present in the sample. The consensus primers may also be suitable in a ligase chain reaction. For example, in PCT Patent Publication No. WO 89/09835, a process is described that involves the use of ligase to join oligonucleotide segments that anneal to the target nucleic acid. This publication is incorporated herein by reference.
The invention, although applicable to any HPV, is exemplified below with reference to genital and dermal HPV types. Furthermore, the primers and probes of the invention can be targeted to areas of the HPV genome other than those described below, provided that the particular area targeted can be amplified using consensus primers and the amplified DNA can be typed using type-specific probes.
The present invention has led and will continue to lead to the discovery of many previously unknown or uncharacterized HPV types. However, with each new HPV type discovered there comes a corresponding need to make the consensus probe more genetic to ensure that the new type will be detected and to make type-specific probes. To overcome this problem, the present invention provides an alternative type of consensus probe. This new type of probe, referred to herein as a long probe, comprises a sequence of DNA from one or more HPV viruses that comprises all or most of the HPV genomic DNA sequence that lies between two HPV amplification primers. Long probes provide enhanced sensitivity and reduce the likelihood of a false negative result even when a novel variant HPV-type is present.
The first step of the method of the present invention involves the amplification of an HPV sequence, if that sequence is present in a sample, by PCR using consensus primers. Illustrative consensus primers of the invention are referred to by the region of the HPV genome the primers are used to amplify. The HPV genome is circular. The genome of genital HPVs is oriented as follows: E6, E7, E1, E2, E4, E5a, E5b, L2, L1, and URR. "E" and "L" designations indicate open reading frames and URR indicates the transcriptional regulatory region. Several of the open reading frames overlap, for example, the E4 region is totally contained within the E2 open reading frame. Primers can be used to amplify a sequence that spans one or more regions of the HPV genome. The methods and compositions described herein are particularly suited for amplifying the following HPV regions: L1/URR, L1, E6, E6/E7, E7 through E1, E6 through E 1, and E1. It will be clear to one of ordinary skill in the art that the methods disclosed are applicable to any region of the HPV genome and are not limited to the specific embodiments described herein. Throughout the specification nucleotides are designated as follows.
______________________________________Symbol Meaning Origin______________________________________G G GuanineA A AdenineT T ThymineC C CytosineR G or A puRineY T or C pYrimidineM A or C aMinoK G or T KetoS G or C Strong interaction (3 H bonds)W A or T Weak interaction (2 H bonds)H A or C or T not-G, H follows G in the alphabetB G or T or C not-A, B follows AV G or C or A not-T (not-U), V follows UD G or A or T not-C, D follows CN G or A or T or C aNy______________________________________
Following amplification, the method of the invention describes that a determination is made as to whether amplification has occurred. There are a variety of known means for determining whether amplification has occurred. For example, a portion of the PCR reaction mixture can be subjected to gel electrophoresis and the resulting gel stained with ethidium bromide and exposed to ultraviolet light to observe whether a product of the expected size is present. Alternatively, labeled PCR primers or deoxyribonucleoside 5'-triphosphates are utilized and incorporation of the label into the amplified DNA is determined by, for example, gel electrophoresis and autoradiograph to ascertain if amplification occurred. Another method for determining if amplification has occurred is to test a portion of PCR reaction mixture for ability to hybridize to a labeled probe designed to hybridize to only the amplified DNA. The probe must be a consensus probe so that amplified DNA from any HPV can be detected.
Alternatively, the determination of amplification and identification of HPV type can be carried out in one step by testing a portion of the PCR reaction mixture for its ability to hybridize to one or more type-specific probes. In another aspect of the invention multiple HPV infections are detected by restriction endonuclease digestion of amplification products. Restriction endonuclease analysis of amplified HPV DNAs is also useful for detecting variants and novel HPV types.
The invention encompasses consensus probes and type-specific probes. The probes may be oligonucleotides that are similar in length to primers or may be long probes. Long probes are especially preferred as the consensus probes for generic detection of amplified HPV nucleic acids.
As used herein "long probes" refer to nucleic acid segments used as generic probes for determining if amplification of any HPV has occurred. Long probes are generally greater than 100 nucleotides in length and may be as long as the amplified fragment they are designed to detect. Preferably, long probes are between 100-600 base pairs in length and comprise the full length of the amplified fragment to be detected, but do not include the primer sequences used to generate the amplified segment. For example, in Example II, the long probes are 410 nucleotides long (450, the L1 amplified product length-2.times.20, the L1 consensus primer length). The long probes are preferably PCR fragments. Most preferably, the PCR primers used to prepare the long probes are nested primers relative to the consensus primers used to amplify the HPV DNA in a sample so that the consensus primer sequences are not included in the probe.
The long probes are used as a mixture suitable for generic detection of HPV nucleic acids, and at least one nucleic acid in the generic long probe mixture will hybridize to each of HPV types -6, -11, -16, - 18, and -33. However, it is not necessary to include amplified nucleic acids from each major HPV type in a long generic probe mixture. In one embodiment, Example I describes that long probes are prepared using HPV-16, HPV 18, and two clinical samples as the PCR template. The example describes a 410 base pair probe mixture that hybridizes to the major HPV types, as well as minor, variant, or novel HPV types. The long probes may be single-stranded or double-stranded. As used herein, long probes are also referred to as "generic probes" or "long generic probes."
Thus an important aspect of the present invention relates to the novel probes provided for use in the present methods. Whether these probes are consensus probes for determining if amplification has occurred or type-specific probes, the probes can be used in a variety of different hybridization formats. Although solution hybridization of a nucleic acid probe to a complementary target sequence is clearly within the scope of the present invention, commercialization of the invention will likely result in the use of immobilized probes and thus a quasi "solid-phase" hybridization. In this format, the probe is covalently attached to a solid support and then the target sequences are then hybridized to the probe. In this method, the probes are attached to a solid support by virtue of long stretches of T residues; these T residues are added to the probe during synthesis of the probe on an automated synthesizer after the hybridizing sequence is synthesized. When long probes are used in the method T-tailing is unnecessary and the probes can be attached directly to the solid support. A variety of dyes and chromogens and corresponding labels are available for nucleic acid detection systems.
In one embodiment, the L1/E6 consensus primer combinations of the invention are designed to amplify a sequence of DNA from any genital HPV. The amplified sequence extends from L1 across the URR and into E6 and thus contains portions of the L1 and E6 regions with the URR region sandwiched in between the L1 and E6 regions. Thus, the consensus primer pairs consist of a first primer specific for a sequence in the L1 region and a second primer specific for a sequence in the E6 region. As shown in Table 1, below, the first L1-specific primer can be either FS10, FS17, or MY01, while the second, E6-specific primer is at least a 1:1 mixture of JS15 and JS16, although the mixture can also contain more JS15 than JS16. Table 1 also depicts the sequence each primer and the corresponding sequence (and nucleotide position of that sequence) as it occurs in the genomes of several well-known genital HPVs (Types 6, 11, 16, 18, and 33). A dash in a sequence indicates that the genomic sequence is identical to the primer sequence.
TABLE 1__________________________________________________________________________L1/E6 Consensus Primers and Amplification ProductsL1 Consensus Positive Strand Primers__________________________________________________________________________FS10 25mer 5' C T G T G G T A G A T A C C A C A C G C A G T A CHPV06 6770 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --HPV11 6756 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --HPV16 6633 -- -- -- -- T -- -- T -- -- -- -- -- T -- -- -- -- -- -- -- -- -- -- --HPV18 6587 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- -- -- -- --HPV33 6587 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- -- -- -- --FS17 20mer 5' G A T C A G T T T C C Y Y T K G G A C GMY01 20mer 5' G A T C A G T W T C C Y Y T K G G A C GHPV06 7151 -- -- -- -- -- -- -- A -- -- -- T T -- G -- -- -- -- --HPV11 7136 -- -- -- -- -- -- -- T -- -- -- C C -- T -- -- -- -- --HPV16 7015 -- -- -- -- -- -- -- T -- -- -- T T -- A -- -- -- -- --HPV18 6993 -- -- -- -- -- A -- A -- -- -- C C -- T -- -- -- -- --HPV33 6968 -- -- -- -- -- -- -- T -- -- -- T T -- G -- -- -- -- --__________________________________________________________________________URR/E6 Consensus Negative Strand Primer__________________________________________________________________________JS15 18mer 5' C C G T T T T C G G T T S A A C C GHPV06 60 -- -- -- -- -- -- -- -- -- -- -- -- G -- -- -- -- --HPV11 60 -- -- -- -- -- -- -- -- -- -- -- -- G -- -- -- -- --HPV16 60 -- -- -- G -- -- -- -- -- -- -- -- C -- -- -- -- --HPV33 64 -- -- -- -- -- -- -- -- -- -- -- -- G -- -- -- -- --JS16 19mer 5' C C G T T T T C G G T C C C G A C C GHPV18 68 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --__________________________________________________________________________Predicted Sizes Of PCR Products__________________________________________________________________________ + PRIMER FS10 FS17 or MYO1 - PRIMER JS15 and JS16 JS15 and JS16 HPV06 1192 bp 822 bp HPV11 1235 bp 856 bp HPV16 1387 bp 958 bp HPV18 1367 bp 932 bp HPV33 1434 bp 1005 bp__________________________________________________________________________
As shown in Table 1, FS 10 is a 25-mer that has 3 mismatches with HPV16 and 1 mismatch with HPV18 and HPV33. FS17 is a degenerate primer with 1 or 2 mismatches to different HPVs. MY01 is similar to FS 17, contains 1 more degenerate base to decrease mismatches and to potentially cover a wider range of HPVs. JS15 is a degenerate 18-mer for the negative strand in E6 of HPVs 6, 11, 16, and 33, whereas JS16 is a 19-mer serving the same function for HPV18.
Once a sample has been treated with the L1/E6 primers shown above under conditions suitable for PCR, the method of the invention requires the determination of whether amplification has occurred. If amplification has occurred with the L1/E6 primers, HPV sequences are present in the sample. In one aspect of the invention, a consensus probe is used to determine if amplification has occurred. Alternatively, amplification of HPV DNA using the L1/E6 consensus primers FS10, JS15, and JS16 can be detected using the L1/E6 consensus primer FS17 or MY01.
The present invention provides a number of type-specific probes for use with the L1/E6 consensus primers of the invention. These probes are set forth in Table 2, below. Those skilled in the art will recognize that although the specific primers and probes of the invention exemplified herein have a defined number of nucleotide residues, one or more nucleotide residues may be added or deleted from a given primer or probe typically without great impact on the suitability of that primer or probe in the present methods.
TABLE 2__________________________________________________________________________HPV Typing Probes For Use with L1/E6 Consensus PrimersSpecificity Sequence Size Designation__________________________________________________________________________HPV6 5'CCAAACAGTAAGAGC (15-mer) FS18HPV11 5'GGCTGTAGAGGGCTTAGAC (19-mer) FS19HPV16 5'GGTTGAAGCTACAAAATGGGCC (22-mer) JS17HPV18 5'GTAGCGCACCTGGACAGG (18-mer) FS21HPV33 5'CAGGTAGTGACTCAC (15-mer) FS22__________________________________________________________________________
FS19 and JS17 can specifically detect HPV11 and HPV16, respectively. FS18 shows some hybridization with the HPV11 PCR product. UWGCG GAP program analysis comparing FS18 sequence and HPV11 sequence indicates a 73% homology of FS18 to HPV11 in the amplified region. The cross-hybridization could be minimized by increasing the stringency of washing. FS21 was specific for HPV18.
The L1/E6 primers disclosed above provide for the amplification of relatively large segments of HPV DNA. However, use of primers that result in shorter PCR products can have several advantages, including reduced extension and denaturation time and decreased denaturation temperature. The L1 consensus primers of the invention are illustrative of primers of the invention designed to amplify relatively small segments of the HPV genome to achieve such advantages. The L1 consensus primers produce a PCR product corresponding to sequences in the L1 open reading frame and are depicted in Table 3, below.
TABLE 3__________________________________________________________________________L1 Consensus Primers and Amplification ProductsL1 Consensus Positive Strand Primer__________________________________________________________________________MY11 20mer 5' G C M C A G G G W C A T A A Y A A T G GHPV06 6722 -- -- C -- -- -- -- -- A -- -- -- -- -- C -- -- -- -- --HPV11 6707 -- -- T -- -- -- -- -- A -- -- -- -- -- C -- -- -- -- --HPV16 6584 -- -- A -- -- -- -- -- C -- -- C -- -- C -- -- -- -- --HPV18 6558 -- -- A -- -- -- -- -- T -- -- -- -- -- C -- -- -- -- --HPV31 -- -- T -- -- -- -- -- A -- -- C -- -- T -- -- -- -- --HPV33 6539 -- -- A -- -- A -- -- T -- -- -- -- -- T -- -- -- -- --__________________________________________________________________________L1 Consensus Negative Strand Primer__________________________________________________________________________MY09 20mer 5' C G T C C M A R R G G A W A C T G A T CHPV06 7171 -- -- -- -- -- C -- A A -- -- -- T -- -- -- -- -- -- --HPV11 7155 -- -- -- -- -- A -- G G -- -- -- A -- -- -- -- -- -- --HPV16 7035 -- -- -- -- -- T -- A A -- -- -- A -- -- -- -- -- -- --HPV18 7012 -- -- -- -- -- A -- G G -- -- -- T -- T -- -- -- -- --HPV31 -- -- A -- -- C -- A A -- -- -- A -- -- -- -- -- -- --HPV33 6988 -- -- -- -- -- C -- A A -- -- -- A -- 1' -- -- -- -- --HPV54 -- -- A -- -- A -- -- G -- -- -- A -- -- -- -- G -- --__________________________________________________________________________Predicted sizes of PCR products from theMY11 and MY09 L1 Consensus Primer Pair__________________________________________________________________________ HPV06 448 bp HPV11 448 bp HPV16 451 bp HPV18 454 bp HPV33 448 bp__________________________________________________________________________
A preferred embodiment of the method of the present invention for genital HPVs comprises amplification of HPV sequences, if present in the sample, with the L1 consensus primers MY09 and MY11; determination of amplification by hybridization of a portion of the PCR reaction mixture with a generic HPV long probe; and finally, type determination with type-specific probes. The invention is useful for detecting any HPV DNA. In a preferred embodiment, which utilizes consensus primers MY09 and MY11, the method is useful for detecting and typing nucleic acids of both dermal and genital HPVs including genital HPV types 6, 6B, 11, 16, 18, 30, 31, 33, 35, 39, 40, 42, 43, 45, 51, 52, 53, 54, 55, 57, 58, and 59, as well as dermal HPV types 5, 8, 26, 27, 41, 47, and 48. At present, more than 60 unique HPV types have been identified. These are briefly described in deVilliers, 1989, J. Virol. 63:4898. The present invention is useful for typing known HPV types as well as identifying unique types or variants. To determine if amplification of HPV DNA sequences has occurred in a sample that has been treated with the L1 consensus primers of the invention, a portion of the PCR reaction mixture can be hybridized with L1 consensus probes, depicted in Table 4.
TABLE 4__________________________________________________________________________L1 Consensus Probes__________________________________________________________________________FS10 5' C T G T G G T A G A T A C C A C A C G C A G T A CMY18 5' -- -- -- -- T -- -- T -- -- -- -- -- T -- -- -- -- -- -- -- -- -- -- --MY19 5' -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- -- -- -- --__________________________________________________________________________Sequence of HPV types in Region of Consensus Probe__________________________________________________________________________HPV6 6771 5' C T G T G G T A G A T A C C A C A C G C A G T A CHPV11 6756 5' -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --HPV16 6631 5' -- -- -- -- T -- -- T -- -- -- -- -- T -- -- -- -- -- -- -- -- -- -- --HPV18 6607 5' -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- -- -- -- --HPV33 6588 5' -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- -- -- -- --Isolate 36 5' -- -- -- -- T -- -- G -- -- -- -- -- T -- -- C A -- A -- -- C -- --Isolate 88 5' -- A -- -- -- -- -- -- -- -- -- -- -- T -- -- T -- -- -- -- -- C -- --__________________________________________________________________________
As noted above, the diversity of HPV types may mandate the use of longer consensus probes that contain almost all of the amplified sequence except that portion corresponding to the primers used in the amplification step of the present method. This diversity in HPV types also demonstrates the need for the type-specific probes provided by the present invention. In a preferred embodiment, the method utilizes a generic probe comprising PCR fragments. The PCR fragments provide long probes for the use as consensus probes for determining if amplification has occurred. According to Example 1, a generic probe was synthesized from the 450 base pair L1 PCR fragments of HPV-16, HPV-18, and the highly divergent isolates PAP88 and PAP238B. The generic probe described in Example 1 comprises segments approximately 400 base pairs in length and can be prepared using the primers depicted below. The primers are used in pairs corresponding to the target; i.e., MY74 (Seq ID No. 1) and MY75 (Seq ID No. 2) for HPV16, MY76 (Seq ID No. 3) and MY77 (Seq ID No. 4) for HPV18, MY47 (Seq ID No. 5) and MY48 (Seq ID No. 6) for PAP88, and MY49 (Seq ID No. 7) and MY50 (Seq ID No. 8) for PAP238B.
__________________________________________________________________________Internal PCR Primers for Generic ProbeName Sequence Target__________________________________________________________________________MY74 (SEQ ID NO. 1) CATTTGTTGGGGTAACCAAC HPV 16MY75 (SEQ ID NO. 2) TAGGTCTGCAGAAAACTTTTC HPV 16MY76 (SEQ ID NO. 3) TGTTTGCTGGCATAATCAAT HPV 18MY77 (SEQ ID NO. 4) TAAGTCTAAAGAAAACTTTTC HPV 18MY47 (SEQ ID NO. 5) CATATGCTGGGGTAATCAGG PAP 88MY48 (SEQ ID NO. 6) CAGGTCTGCAGAAAAGCTGT PAP 88MY49 (SEQ ID NO. 7) TATTTGTTGGGGCAATCAG PAP 238BMY50 (SEQ ID NO. 8) CTAAATCTGCAGAAAACTTTT PAP 238B__________________________________________________________________________
When a portion of the PCR reaction mixture contains DNA that hybridizes to a probe contained in the L1 consensus probe mixture, the sample contains HPV DNA. There are a number of ways to determine whether a probe has hybridized to a DNA sequence contained in a sample. Typically, the probe is labeled in a detectable manner, the target DNA (i.e., the amplified DNA in the PCR reaction buffer) is bound to a solid support, and determination of whether hybridization has occurred simply involves determining whether the label is present on the solid support. This procedure can be varied, however, and is possible when the target is labeled and the probe is bound to the solid support.
Many methods for labelling nucleic acids, whether probe or target, are known in the art and are suitable for purposes of the present invention. Suitable labels may provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Suitable labels include fluoruphores, chromophores, radioactive isotopes (particularly .sup.32 P and .sup.125 I), electrondense reagents, enzymes and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horse-radish-peroxidase (HRP) can be detected by its ability to convert diaminobenzidine to a blue pigment. A preferred method for HRP based detection uses tetramethyl-benzidine (TMB) as described in Clin. Chem. 33:1368 (1987). An alternative detection system is the Enhanced Chemiluminescent (ECL) detection kit commercially available from Amersham. The kit is used in accordance with the manufacturer's directions.
Probes are typically labeled with radioactive phosphorous .sup.32 P, by treating the probes with polynucleotide kinase in the presence of radiolabeled ATP. However, for commercial purposes non-radioactive labeling systems may be preferred, such as, horseradish peroxidase-avidin-biotin or alkaline phosphatase detection systems. HRP can be used in a number of ways. For example, if the primer or one or more of the dNTPs utilized in a PCR amplification has been labeled (for instance, the biotinylated dUTP derivatives described by Lo et al., 1988, Nuc. Acids Res. 16:8719) instead of the probe, then hybridization can be detected by assay for the presence of labeled PCR product. In a preferred embodiment, probes are biotinylated and detected with the ECL system described above. For example, biotinylated probes were prepared by direct biotinylation of the oligonucleotide rather than incorporation of biotin-dUTP during PCR. For 5' biotinylation of oligonucleotides direct solid phrase synthesis using biotin containing phosphoramidites was done according to Alves el al., 1989, Tetra. Let 30:3098; Cocuzza, 1989, Tetra Let. 30:6287; and Barabino et al., 1989, EMBO J. 8:4171. Solid phase synthesis of biotinylated oligonucleotides at any internal or terminal (5' or 3') position is also suitable for preparing biotinylated primers and probes (Pieles et al., 1989, NAR 18:4355, and Misiura et al., 1989, NAR 18:4345).
Alternatively, biotinylated probes can be prepared using terminal deoxynucleotide transferase (TdT) (Boeringer Mannheim) as follows. An 8:1 molar ratio of biotin-dUTP: oligonucleotide provided 80% derivatization with each molecule bearing 1 or more terminal biotin dUMPs. The tailing reaction contained 100 pmoles of oligonucleotide and 800 pmoles of biotin-dUTP in a 10-20 .mu.l reaction using 3-10 units of TdT for 30-60 minutes at 37.degree. C. The reaction buffer was prepared according to the enzyme specifications from the manufacturer. The reaction was stopped with EDTA and purified from 2.5 M NH.sub.4 Ac, by ethanol precipitation (twice) using 10 .mu.g tRNA, ssDNA, glycogen or linearized acrylamide as a carder (see Focus 11(3):57-58). Tailed oligonuclcotides can be probes for targets fixed to a solid support. The Amersham ECL system provides an appropriate means for detecting these compounds (see Nature 346:297[1990]).
In one embodiment of the invention biotinylated probes are prepared by incorporating biotin-dUTP into the amplification reaction for preparing long probes.
Alternatively, probes are conjugated to HRP, for example, according to the method disclosed in WO 89/2932, and Beaucage et al., 1981, Tetra. Lett. 22:1859-1862. These references are incorporated herein by reference. For HRP conjugated probes, the hybridization buffer is adjusted to decrease the wash temperature required for removing non-specifically hybridized probe without the risk of denaturing the HRP enzyme. However, HRP conjugated probes are not used in the preferred embodiment. Whatever the labeling system used, once a determination has been made that the L1 consensus probe has hybridized to amplified DNA present in the sample, the amplified DNA is typed to determine the HPV types present in the sample.
Practice of the present invention has led to the discovery of many previously uncharacterized HPV types. For example, three clinical samples examined by the present method contained five different HPV types with sequences markedly different than the published sequences for HPVs. These new sequences are an important aspect of the present invention, as are the probes that will hybridize to these sequences in a type-specific fashion. These new sequences are depicted below. Degenerate nucleotides are as defined above and correspond to the degenerate nucleotides in the primers used to amplify the region or to variation within the type.
__________________________________________________________________________The DNA Sequence of the L1 Amplified Regions of HPVIsolates 36A, 36B, 88, 238A, and 238B, 155, C14__________________________________________________________________________Isolate 36A 1 GCMCAGGGWC ATAAYAATGG TATATGTTGG CACAATCAAT TGTTTTTAAC 51 AGTTGTAGAT ACTACTCGCA GCACCAATCT YTCTGTGTGT GCTTCTACTA101 CTTCTCCTAT TCCTAATGAA TACACACCTA CCAGTTTTAA AGAATATGCC151 AGACATGTGG RGGAATTTGA TTTGCAGTTT ATAYTTCAAC TGTGTAAAAT201 AACWTTAACT ACAGAGGTAA TGTCATACAT TCATAATATG AATACCACTA251 TTTTGGAGGA TTGGAATTTT GGTRTTACAC CACCTCCTAC TGCTARTTTA301 GTTGACACAT ACCGTTTTGT TCAATCTGCT GCTGTAACTT GTCAAAAGGA351 CACCGCACCG CCAGTTAAAC AGGACCCTTA TGACAAACTA AAGTTTTGGA401 CTGTAAATCT TAAGGAAAGG TTTTCTGCAG ATCTTGATCA GTWTCCYYTK451 GGACGIsolate 36B 1 GCMCAGGGWC ATAAYAATGG TATATGTTGG GGAAATCAGC TATTTTTAAC 51 TGTGGTTGAT ACTACCCGTA GTACTAACAT GACTTTGTGY GCCACTGCAA101 CATCTGGTGA TACATATACA GCTGCTAATT TTAAGGAATA TTTAAGACAT151 GCTGAAGAAT ATGATGTGCA ATTTATATTT CAATTGTGTR AAATAACATT201 AACTGTTGAA GTTATGTCAT ATATACACAA TATGAATCCT AACATATTAG251 AGGAGTGGAA TGTTGGTGTT GCACCACCAC CTTCAGGAAC TTTAGAAGAT301 AGTTATAGGT ATGTACAATC AGAAGCTATT CGCTGTCAGG CTAAGGTAAC351 AACGCCAGAA AAAAAGGATC CTTATTCAGA CTTTTCCTTT TGGGAGGTAA401 ATTTATCTGA AAAGTTTTCT ACTGATTTAG GATCAGTWTC CYYTKGGACGIsolate 88 1 GCMCAGGGWC ATAAYAATGG CATATGCTGG GGTAATCAGG TATTTGTTAC 51 TGTTGTGGAT ACTACCAGAA GCACCAACAT GACTATTAAT GCAGCTAAAA101 GCACATTARC TAAATATGAT GCCCGTGAAA TCAATCAATA CCTTCGCCAT151 GTGGAGGAAT ATGAACTACA GTTTGTGTTT CAACTTTGTA AAATAACCTT201 AACTGCAGAR GTTATGGCAT ATTTGCATAA TATGAATAAT ACTTTATTRG251 ACGATTGGAA TATTGGCTTA TCCCCACCAG TTGCAACTAG CTTAGAGGAT301 AAATATAGGT ATATTAAAAG CACAGCTRTT ACAYGTCAGA GGGAACAGCC351 CCCTGCAGAA AAGCAGGATC CCCTGGCTAA ATATAAGTTT TGGGAAGTTA401 ATTTACAGGA CAGCTTTTCT GCAGACCTGG GATCAGTWTC CYYTKGGACGIsolate 238A 1 GCMCAGGGWC ATAAYAATGG TATTTGTTGG VATAATCART TATTTTTAAC 51 TGTTGTAGAT ACTACTAGAA GCACTAATTT TTCTGTATGT GTAGGTACAC101 AGGCTAGTAG CTCTACTACA ACGTATGCCA ACTCTAATTT TAAGGAATAT151 TTAAGACATG CAGAAGAGTT TGATTTACAG TTTGTTYTTC AGTTATGTAA201 AATTAGTTTA ACTACTGAGG TAATGACATA TATACATTCT ATGAATTCTA251 CTATATTGGA AGAGTGGAAT TTTGGTCTTA CCCCACCACC GTCAGGTACT301 TTAGAGGAAA CATATAGATA TGTAACATCA CAKGCTATTA GTTGCCAACG351 TCCTCAACCT CCTAAAGAAA CAGAGGACCC ATATGCCAAG CTATCCTTTT401 GGGATGTAGA TCTTAAGGAA AAGTTTTCTG CAGAATTAGA TCAGTWTCCY451 YTKGGACGIsolate 238B 1 GCMCAGGGWC ATAAYAATGG TATTTGTTGG GGCAATCAGT TATTTGTTAC 51 TGTGGTAGAT ACCACACGTA GTACCAATAT GTCTGTGTGT GCTGCAATTG101 CAAACAGTGA TACTACATTT AAAAGTAGTA ATTTTAAAGA GTATTTAAGA151 CATGGTGAGG AATTTGATTT ACRATTTATA TTTCAGTTAT GCAAAATAAC201 ATTATCTGCA GACATAATGA CATATATTCA CAGTATGAAT CCTGCTATTT251 TGGAAGATTG GAATTTTGGA TTGACCACAC CTCCCTCAGG TTCTTTAGAG301 GATACCTATA GGTTTGTAAC CTCACAGGCC ATTACATGTC AAAAARCTGC351 CCCCCAAAAG CCCAAGGAAG ATCCATTTAA AGATTATGTA TTTTGGGAGG401 TTAATTTAAA AGAAAAGTTT TCTGCAGATT TAGATCAGTW TCCYYTKGGA451 CGIsolate 155A and 155B 1 TATATGCTGG TTTAATCAAT TGTTTGTCAC GGTGGTGGAT ACCACCCGCA 51 GCACCAATTT TACTATTAGT GCTGCTACCA ACACCGAATC AGAATATAAA101 CCTACCAATT TTAAGGAATA CCTAAGACAT GTGGAGGAAT ATGATTTGCA151 GTTTATATTC CAGTTGTGTA AGGTCCGTCT GACTCCAGAG GTCATGTCCT201 ATTTACATAC TATGAATGAC TCCTTATTAG ATGAGTGGAA TTTTGGTGTT251 GTGCCCCCTC CCTCCACAAG TTTAGATGAT ACCTATAGGT ACTTGCAGTC301 TCGCGCCATT ACTTGCCAAA AGGGGGCCGC CGCCGCCAAG CCTAAGGAAG351 ATCCTTATGC TGGCATGTCC TTTTGGGATG TAGATTTAAA GGACAAGTTT401 TCTACTGATT TGIsolate C14 1 TATTTGTTGG CATAATCAGT TGTTTGTTAC TGTAGTGGAC ACTACCCGCA 51 GTACTAATTT AACATTATGT GCCCTCTACAC AAAATCCTGT GCCAAATACA101 TATGATCCTA CTAAGTTTAA GCACTATAGT AGACATGTGG AGGAATATGA151 TTTACAGTTT ATTTTTCAGT TGTGCACTAT TACTTTAACT GCAGAGGTTA201 TGCCATATAT CCATAGTATG AATAGTAGTA TATTGGAAAA TTGGAATTTT251 GGTGTACCTC CACCACCTAC TACAAGTTTA GTGGATACAT ATCGTTTTGT301 GCAATCCGTT GCTGTTACCT GTCAAAAGGA TACTACACCT CCAGAAAAGC351 AGGATCCATA TGATAAATTA AAGTTTTGGA CTGTTGACCT AAAGGAAAAA401 TTTTCCTCCG ATTTG__________________________________________________________________________
Those skilled in the art will recognize that with the above sequence information, primers and probes for amplifying and detecting these new HPV isolates can be readily obtained. In addition, these sequences enable one to isolate the entire virus from samples containing the virus. Isolate 36A is an HPV59 variant, and isolate 36B is an HPV42 variant. Isolate 238B corresponds to the HPV31 type described in the literature. A cervical carcinoma isolate, C14, is a variant of HPV45. The discovery of these new HPV isolates led to the creation of additional L1 consensus oligonucleotide probes for use in conjunction with FS10, MY18, and MY19. These L1 consensus probes are depicted below under the FS 10 probe sequence to demonstrate the similarity to the FS10 sequence. L1 consensus probe WD147 will hybridize to HPV45 DNA. MY46 (Seq ID No. 9) is a combination of FS10 and MY19. In one embodiment, a consensus L1 probe can be an equimolar mixture of MY 18, MY46, (Seq ID No. 9) MY57, and WD 147.
__________________________________________________________________________FS10 5' C T G T G G T A G A T A C C A C A C G C A G T A CMY66 -- A -- -- T -- -- -- -- -- -- -- -- T -- -- T -- -- -- -- -- C -- --MY55 -- -- -- -- -- -- -- T -- -- -- -- -- T -- -- C -- -- T -- -- -- -- --MY39 -- -- -- -- T -- -- G -- -- -- -- -- T -- -- C A -- A -- -- C -- --MY56 -- -- -- -- T -- -- -- -- -- -- -- -- T -- -- T A -- A -- -- C -- --MY57 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- --WD147 -- -- -- -- A -- -- G -- -- C -- -- T -- -- C -- -- -- -- -- -- -- --MY46 (SEQ -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- W -- -- -- -- -- -- -- --ID NO. 9)__________________________________________________________________________
The present invention provides a number of probes for typing amplified DNA produced from L1 consensus primers. While most probes for typing HPV hybridize to known HPV types, others specifically detect previously uncharacterized HPV types and hybridize to novel HPVs identified in clinical samples as a result of use of the present methods. These probes are depicted below in Table 5.
TABLE 5__________________________________________________________________________HPV Typing Probes For Use with L1 Consensus Primers Genome 5' BiotinylatedProbe Specificity Sense Sequence Position Versions__________________________________________________________________________MY12 HPV6 + CATCCGTAACTACATCTTCCA 6813 MY134MY62 HPV6 + CTCCTGAAAAGGAAAAGCCA 7068MY63 HPV6 - TGGCTTTTCCTTTTCAGGAG 7068 MY135MY125 (SEQ HPV6/HPV11 + ACAATGAATCYTCTGTTTTGG 6950/6935 MY138ID NO. 10)MY126 (SEQ HPV6/HPV11 + ATCGCCTCCMCCAAATG 6991/6976ID NO. 11)MY13 HPV11 + TCTGTGTCTAAATCTGCTACA 6800 MY136MY61 HPV11 + CACACCTGAAAAAGAAAAACAG 7051MY65 HPV11 - CTGTTTTTCTTTTTCAGGTGTG 7051 MY137WD150 HPV11 + CAGAAACCCACACCTGAAAAAGA 7059WD151 HPV11 + AGAAACCCACACCTGAAAAAGAA 7058MY14 HPV16 + CATACACCTCCAGCACCTAA 6926 MY14MY58 HPV16 + TTGTAACCCAGGCAATTGCT 6897MY71 (SEQ HPV16 + ACATACACCTCCAGCACCTA 6925ID NO. 12)MY72 (SEQ HPV16 + CATACACCTCCAGCACCTA 6926ID NO. 13)MY95 (SEQ HPV16 - GATATGGCAGCACATAATGAC 6685 MY139ID NO. 14)MY96 (SEQ HPV16 - AGTTTCTGAAGTAGATATGGCA 6698ID NO. 15)MY97 (SEQ HPV16 - CTGAAGTAGATATGGCAGCAC 6693ID NO. 16)MY133 (SEQ HPV16 + GTAACATCCCAGGCAATTG 6897 MY140ID NO. 17)WD152 HPV16 + TTTGTAACCCAGGCAATTGCT 6898WD153 HPV16 + GTTGTAACCCAGGCAATTGCT 6897MY60 HPV18 + CAGTCTCCTGTACCTGGG 6657MY73 (SEQ HPV18 + GATGCTGCACCGGCTGAA 6906ID NO. 18)MY107 (SEQ HPV18 - GCCCAGGTACAGGAGAC 6659ID NO. 19)MY130 (SEQ HPV18 + GGGCAATATGATGCTACCAAT 6672 MY142ID NO. 20)MY131 (SEQ HPV18 + GTACCTGGGCAATATGATG 6666ID NO. 21)MY132 (SEQ HPV18 + TCTCCTGTACCTGGGCAA 6660ID NO. 22)WD74 HPV18 + GGATGCTGCACCGGCTGA 6905 MY141WD75 (SEQ HPV18 - TCAGCCGGTGCAGCATCC 6922ID NO. 23)WD126 HPV31 + CCAAAAGCCCAAGGAAGATC 6851WD127 HPV31 + CAAAAGCCCAAGGAAGATC 6852WD128 HPV31 + TTGCAAACAGTGATACTACATT 6597 MY143MY109 (SEQ HPV31 - GAGGGAGGTGTGGTCAAT 6769ID NO. 24)MY110 (SEQ HPV31 - AAGAACCTGAGGGAGGT 6778ID NO. 25)MY92 (SEQ HPV31/HPV31B + CCAAAAGCCYAAGGAAGATC 6853 MY144ID NO. 26)MY127 (SEQ HPV31/HPV31B + ACCACACCTCCCTCAG 6773ID NO. 27)MY128 (SEQ HPV31/HPV31B + ACAGGCCATTACATGTCAA 6823ID NO. 28)MY16 HPV33 + CACACAAGTAACTAGTGACAG 6628 MY145MY59 HPV33 + AAAAACAGTACCTCCAAAGGA 6877MY64 HPV33 - TCCTTTGGAGGTACTGTTTTT 6877 MY146MY115 (SEQ HPV35 + CTGCTGTGTCTTCTAGTGACAGID NO. 29)MY116 (SEQ HPV35 + TGCACCAAAACCTAAAGATGID NO. 30)MY117 (SEQ HPV35 - ATCATCTTTAGGTTTTGGTGCID NO. 31)MY89 (SEQ HPV39 + TAGAGTCTTCCATACCTTCTAC MY147ID NO. 32)MY90 (SEQ HPV39 + CTGTAGCTCCTCCACCATCT MY148ID NO. 33)MY91 (SEQ HPV39 + AGACACTTACAGATACCTACAGID NO. 34)MY118 (SEQ HPV40 + CACACCAGGCCCATATAATID NO. 35)MY119 (SEQ HPV40 + CCAAGGTACGGGAGGATCID NO. 36)MY120 (SEQ HPV40 - GATCCTCCCGTACCTTGID NO. 37)MY121 (SEQ HPV42 + CACTGCAACATCTGGTGATID NO. 38)MY122 (SEQ HPV42 - TCACCAGATGTTGCAGTGID NO. 39)MY33 (SEQ HPV42 - GGCGTTGTTACCTTAGCCID NO. 40)MY34 (SEQ HPV42 + GGCTAAGGTAACAACGCCID NO. 41)MY68 (SEQ HPV45 + GGATACTACACCTCCAGID NO. 42)MY69 HPV45 + ATACTACACCTCCAGAAAAGC MY149MY70 HPV45 + TAGTGGACACTACCCGCAGMY98 (SEQ) HPV45 - GCACAGGATTTTGTGTAGAGGID NO. 43)MY99 (SEQ HPV45 - TGTATTTGGCACAGGATTTTGID NO. 44)MY100 (SEQ HPV45 - CAGGATTTTGTGTAGAGGCAID NO. 45)MY108 (SEQ HPV45 + CAAATCCTGTGCCAGGTACID NO. 46)MY129 (SEQ HPV45 - GCACAGGATTTTGTGTAGAG MY150ID NO. 47)MY87 (SEQ HPV51 + TATTAGCACTGCCACTGCTGID NO. 48)MY88 (SEQ HPV51 + CCCAACATTTACTCCAAGTAACID NO. 49)MY80 (SEQ HPV52 + CTGAGGTTAGAAAGGAAAGCAID NO. 50)MY81 (SEQ HPV52 + CACTTCTACTGCTATAACTTGTID NO. 51)MY82 (SEQ HPV52 + ACACACCACCTAAAGGAAAGGID NO. 52)MY101 (SEQ HPV53 + CGCAACCACACAGTCTATGTID NO. 53)MY102 (SEQ HPV53 + TTCTACCTTACTGGAAGACTGGID NO. 54)MY103 (SEQ HPV53 + GGAGGTCAATTTGCAAAACID NO. 55)MY111 (SEQ HPV54 - TGCAGGGGCATTATTCTTTID NO. 56)MY112 (SEQ HPV54 + TACAGCATCCACGCAGID NO. 57)MY113 (SEQ HPV54 + CACGCAGGATAGCTTID NO. 58)MY114 (SEQ HPV54 + CCACGCAGGATAGCTTID NO. 59)MY151 (SEQ HPV55 + GTGCTGCTACAACTCAGTCTID NO. 60)MY152 (SEQ HPV55 + GCTACAACTCAGTCTCCATCID NO. 61)MY153 (SEQ HPV55 - TGCCTTTTCAGGGGGAGID NO. 62)MY154 (SEQ HPV57 + AATGTCTCTTTGTGTGCCACID NO. 63)MY155 (SEQ HPV57 + GTGTGCCACTGTAACCACAID NO. 64)MY156 (SEQ HPV57 - GGATCAGTAGGGGTCTTAGGID NO. 65)MY123 (SEQ HPV59 + GCCAGTTAAACAGGACCCID NO. 66)MY124 (SEQ HPV59 - CATAAGGGTCCTGTTTTAACTGID NO. 67)MY83 (SEQ PAP88 + ATTAATGCAGCTAAAAGCACATTID NO. 68)MY84 (SEQ PAP88 + GATGCCCGTGAAATCAATCAAID NO. 69)MY86 (SEQ PAP155 + TACTTGCAGTCTCGCGCCAID NO. 70)MY85 (SEQ PAP155 + CCAACACCGAATCAGAATATAAAID NO. 71)MY93 (SEQ PAP251 + GCACTGAAGTAACTAAGGAAGGID NO. 72)MY94 (SEQ PAP251 + AGCACCCCCTAAAGAAAAGGAID NO. 73)MY104 (SEQ PAP238A + GTAGGTACACAGGCTAGTAGCTCID NO. 74)MY105 (SEQ PAP238A + GCTCTACTACAACGTATGCCAID NO. 75)MY106 (SEQ PAP238A + AGTTGCCAACGTCCTCAACID NO. 76)__________________________________________________________________________
The present invention also provides consensus primers and HPV typing probes for detection of DNA sequences specific for the E6 region of genital HPVs. These probes are particularly preferred, because in some cancers, the HPV genome is partially deleted or rearranged such that only E6- and E7-related sequences are present. The E6 consensus primer pairs of the invention comprise primer pairs in which one primer is complementary to sequences near the border of the URR and E6 regions and the other primer is complementary to sequences in either the E7 region near the E6-E7 border (the E6 and E7 open reading frames overlap) or in the E6 region. These E6 consensus primers are depicted below in Table 6.
Although E6 consensus primers, consensus probes, and type specific probes are suitable for detecting and typing HPV nucleic acids, they also have utility when used in conjunction with L1 primers and probes. In one embodiment of the invention, the E6 gene was chosen as a site for a second consensus primer set and type specific probes. The use of E6 as a second site for detection and typing may allow detection of such integrated HPVs. The smaller E6 PCR products also increase the likelihood of detecting HPV sequences in archival tissues where DNA may be too fragmented to serve as a template for long PCR products.
While it is not an essential aspect of the present invention, the use of two sites allows rapid confirmation of typing results, thereby increasing the confidence level of the data. The use of a second site also circumvents the prospect of false negativity as a consequence of sequence variation in one of the primer binding sites.
TABLE 6__________________________________________________________________________E6 Consensus PrimersURR/E6 Consensus Positive Strand Primers__________________________________________________________________________WD73 5' C G G T T S A A C C G A A A A C G GWD72 5' C G G T C G G G A C C G A A A A C G GWD76 5' C G G T T S A A C C G A A A M C G GWD77 5' C G G T T C A A C C G A A A M C G GHPV6 43 5' C G G T T C A A C C G A A A A C G GHPV11 43 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --HPV16 43 -- -- -- -- -- G -- -- -- -- -- -- -- -- C -- -- --HPV18 43 -- -- -- -- C G G G -- -- -- -- -- -- -- -- -- -- --HPV31 45 G T -- G -- G -- -- -- -- -- -- -- -- -- -- -- --HPV33 65 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --__________________________________________________________________________ The URR/E6 positive strand primers are used as mixtures: WD72 and WD73; WD72 and WD76; and WD72 and WD77.
E7 Consensus Negative Strand Primer__________________________________________________________________________WD70 5' G C R C A G A T G G G R C A C A CWD71 5' G C A C A C C A C G G A C A C A CHPV06 813 5' G C G C A G A T G G G A C A C A CHPV11 813 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --HPV16 845 -- A A -- -- -- -- -- -- -- -- G -- -- -- -- --HPV18 894 -- -- A -- -- C C A C -- -- -- -- -- -- -- --HPV33 856 -- -- A -- -- -- G -- A -- -- G -- -- -- -- --WD68 5' C A C A C A A T D Y Y Y A G T G T G C C CWD69 5' C A C A C A A A G G A C A G G G T G T T CHPV06 801 5' C A C A C T A T G T T T A G T G T T C C CHPV11 801 -- -- -- -- -- A -- -- A -- -- -- -- -- -- -- -- G -- -- --HPV16 833 -- -- -- -- -- A -- -- T C C -- -- -- -- -- -- G -- -- --HPV18 882 -- -- -- -- -- A -- A -- G A C -- -- G -- G T T --HPV33 844 -- -- -- -- -- A -- -- A -- -- C -- C -- -- -- G -- -- --__________________________________________________________________________ The E7 negative strand primers are used as mixtures: WD70 and WD71; and WD68 and WD69.
E6 Consensus Negative Strand Primer__________________________________________________________________________WD67 W G C A W A T G G A W W G C Y G T C T CWD66 A G C A T G C G G T A T A C T G T C T CWD154 T C C G T G T G G T G T G T C G T C C CWD155 W S C A W A T G G W W W G Y C G T C Y CWD163 W G C A W A T G G A W W G Y Y G T C Y CWD164 W S C A W A T G G W D W G Y Y G T C Y CHPV6 286 T G C A T A T G G A T A G C C G C C T CHPV11 286 -- -- -- -- A -- G -- -- -- A -- -- T T -- T -- -- --HPV16 286 A -- -- -- -- -- -- -- -- -- T T -- -- -- A T -- -- --HPV18 292 A -- -- -- -- G C -- -- T A T A -- T -- T -- -- --HPV31 -- C -- G -- G -- -- -- T G T -- T -- -- T -- C --HPV33 390 -- C -- -- A -- -- -- -- -- -- T T -- -- C T -- -- --HPV43 A -- -- A A -- C -- -- -- -- -- T -- -- -- T -- G --HPV44 -- -- -- -- A -- T -- -- -- A -- -- T T T -- -- -- --HPV56 -- -- -- -- -- -- A -- -- -- A -- A T -- A T -- C --__________________________________________________________________________ The E6 negative strand primers are used as mixtures; WD66, WD67 and WD155 or WD66, WD67, AND WD163; WD66, WD67, AND WD164, or most preferably, WD66 WD67, and WD154.
The negative strand primer WD154 was designed to hybridize to HPV31 DNA. Amplification of E6 sequences from HPV31 nucleic acids is improved when WD154 is included. Additional E6 consensus negative strand primers located in a different region of the gene, .about.50 bp distal from WD66, WD67, and WD154, and are used in pairs as shown below to yield a 300 bp product. The three primer sets shown, WD157/WD160; WD158/WD161; and WD159/WD162, each correspond to the same genomic HPV region, but they differ in length and are HPV18 specific.
______________________________________WD157 TTCTAMTGTWGTTSCATAYACASHATAWD160 CCAATGTGTCTCCATACACAGAGTCWD158 CTAMTGTWGTTSCATAYACASHATAWD161 AATGTGTCTCCATACACAGAGTCWD159 TAMTGTWGTTSCATAYACASHATAWD162 ATGTGTCTCCATACACAGAGTC______________________________________
__________________________________________________________________________Predicted Sizes of Products from E6 Consensus Primers Product Size for HPV TypeURR/E6 Primer Downstream Primer HPV6 HPV11 HPV16 HPV18 HPV33__________________________________________________________________________WD72 and WD73 WD70 and WD71 770 770 802 844 791WD72 and WD76 WD70 and WD71 770 770 802 844 791WD72 and WD77 WD70 and WD71 770 770 802 844 791WD72 and WD73 WD68 and WD69 758 758 790 832 779WD72 and WD76 WD68 and WD69 758 758 790 832 779WD72 and WD77 WD68 and WD69 758 758 790 832 779WD72 and WD73 WD66 and WD67 243 243 242 242 225WD72 and WD76 WD66 and WD67 243 243 242 242 225WD72 and WD77 WD66 and WD67 243 243 242 242 225WD72 and WD76 WD66, WD67, and WD154 243 243 242 242 225__________________________________________________________________________
Those skilled in the art will recognize that the E6 consensus primers of the invention amplify a sequence that comprises a portion of E7 DNA. To determine if amplification has occurred when the E6 consensus primers are used in the method of the invention, E6 consensus probes are provided. E6 consensus probes WD136 and WD135 are positive strand oligonucleotides directed to the small E6 amplification product and are used together as a mixture. The E6 consensus probes can also be used as E6 consensus positive strand primers. WD135 will hybridize to HPV6 and HPV11 DNA. WD136 is complementary to HPV types 16, 18, 33, and 39. When used as E6 consensus primers, the E6 consensus probes are used in the following combinations: WD65 and WD64; WD83 and WD64; and WD84 and WD64. The E6 consensus probes to two regions are depicted in Table 7.
TABLE 7__________________________________________________________________________E6 Consensus Probes for E6/E7 Product__________________________________________________________________________WD65 M G A G A C R G C W W T C C A T W T GWD83 M G A G A C R G S W W T C C A T W T GWD84 M G A G A C R G V W W T C C A T W T GWD64 A G A G A C A G T A T A C C G C A T GHPV6 267 C G A G G C G G C T A T C C A T A T GHPV11 267 -- -- -- -- A -- A A -- -- T -- -- -- C -- T -- --HPV16 266 A -- -- -- A T -- 1' G A -- -- -- -- -- -- -- -- --HPV18 273 A -- -- -- A -- A -- T A T A -- -- G C -- --HPV33 271 A -- -- -- A G -- -- A A -- -- -- -- -- -- T -- --__________________________________________________________________________Consensus Probes for Detection of Small E6 Product__________________________________________________________________________MY135 G A G A T W T A T K C A T A T G C(SEQIDNO.77)HPV6 225 G A G A T T T A T T C A T A T G CHPV11 225 -- -- -- -- -- A -- -- -- G -- -- -- -- -- -- --MY136 G A G G T A T W T G A H T T T G C(SEQIDNO.78)HPV16 224 G A G G T A T W T G A H T T T G CHPV18 231 -- -- -- -- -- -- -- T -- -- -- A -- -- -- -- --HPV33 229 -- -- -- -- -- -- -- -- -- -- -- T -- -- -- -- --HPV39 -- -- -- -- -- -- -- -- -- -- -- A -- -- -- -- --HPV45 -- -- -- -- -- -- -- -- -- -- -- A -- -- -- -- --__________________________________________________________________________
To determine the type of the HPV present in a sample when the E6 consensus primers are used in the method of the invention, E6 type-specific probes are provided. These probes are depicted in Table 8. Using URR/E6 consensus primers comprising positive strand primers of Table 6 with WD70 and WD71 or WD68 and WD69 any of the HPV typing probes of Table 8 or Table 8A will be effective. These typing probes are also useful when E1 negative strand primers are used for amplification with the URR/E6 positive strand consensus primers.
TABLE 8__________________________________________________________________________HPV Typing Probes for Use with E6/E7 Amplified SequencesProbe Specificity Sequence Genome Position__________________________________________________________________________WD78 HPV6 5' CGAAGTGGACGGACAAGAT 643WD79 HPV11 5' CAAGGTGGACAAACAAGACG 643WD80 HPV16 5' GAACACGTAGAGAAACCCAG 534WD81 HPV18 5' CAACCGAGCACGACAGGA 530WD82 HPV33 5' GAGGTCCCGACGTAGAGAA 534__________________________________________________________________________
TABLE 8A__________________________________________________________________________HPV Typing Probes for Use with Small E6 Amplified SequencesProbe Specificity Sequence Genome Position__________________________________________________________________________WD165 HPV31 5' AAATCCTGCAGAAAGACCTC 116WD166 HPV31 5' CCTACAGACGCCATGTTCAWD167 HPV39 5' CCTTGCAGGACATTACAATAGWD168 HPV39 5' CAGACGACCACTACAGCAAWD169 HPV42 5' GGTGCAAAAAGCATTAACAGWD102 HPV18 5' ACAGTATTGGAACTTACAG 213WD103 HPV16 5' CAACAGTTACTGCGACG 206WD104 HPV33 5' GCAGTAAGGTACTGCAC 88WD132 HPV18 5' GACAGTATTGGAACTTACAG 213WD133 HPV6 5' ACACCTAAAGGTCCTGTTTC 248WD134 HPV11 5' ACACTCTGCAAATTCAGTGC 175RR1 HPV33 5' GTACTGCACGACTATGT 96(SEQIDNO.79)WD171 HPV45 5' ACAAGACGTATCTATTG(SEQIDNO.80)WD170 HPV18 5' GCAAGACATAGAAATAA 178(SEQIDNO.81)RR2 HPV33 5' ACCTTTGCAACGATCTG 212(SEQIDNO.82)__________________________________________________________________________
The typing probes of Table 8A are useful with the positive strand URR, E6 consensus primers WD76/WD72 used with WD67, WD66 and WD 154; WD157 and WD160; WD158 and WD161; WD159 and WD162; WD66 and WD155; WD66 and WD163; and WD66 and WD164. These primers produce a small E6 amplification product of approximately 250 base pairs in length.
The present invention also provides primers that are complementary to sequences in the HPV E1 region. These E1 primers can be used to amplify only E1 region sequences or can be used in conjunction with E6/E7 primers to amplify sequences from E6, E7, E 1, and combinations of these three regions. These E 1 primers are shown below in Table 9.
TABLE 9__________________________________________________________________________E1 POSITIVE STRAND PRIMERS__________________________________________________________________________TYP01 20MER A T G G C K G A Y C C T G M A G G T A CTYP02 20MER -- -- -- -- -- -- -- -- -- G A T T C -- -- -- -- -- --TYP03 20MER -- -- -- -- -- -- -- -- -- C C T T C -- -- -- -- -- --TYP04 20MER T G T A M W G G M T G G T T T T A T G TTYP05 20MER -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- G A G -- --TYP06 20MER -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- A T G -- --__________________________________________________________________________E1 NEGATIVE STRAND PRIMERS__________________________________________________________________________TYN01 20MER G T A C C T K C A G G R T C M G C C A TTYN02 20MER -- -- -- -- -- -- G A A T C -- -- -- -- -- -- -- -- --TYN03 20MER -- -- -- -- -- -- G A A G G -- -- -- -- -- -- -- -- --TYN04 20MER A C A T A A A A C C A K C C W K T A C ATYN05 20MER -- -- C T C -- -- -- -- -- -- -- -- -- -- -- -- -- -- --TYN06 20MER -- -- C A T -- -- -- -- -- -- -- -- -- -- -- -- -- -- --TYN07 20MER T C C A C T T C A G W A T T G C C A T ATYN08 20MER -- -- -- -- -- -- -- -- -- -- -- -- Y A -- -- -- -- -- --__________________________________________________________________________
These E1 primers can be used in a variety of embodiments of the present invention. For instance, amplifications wholly within the E1 region can be performed using the primer pairs: (1) TYP01, TYP02, TYP03, and TYN07, TYN08; or (2) TYP04, TYP05, TYP06 and TYN07, TYN08. Note that TYP03 is similar to both TYP02 and TYP01 and can be omitted from the amplification. The E1 region is highly conserved among HPVs, however, and although typing of the sample is possible with an E1 amplification, typing is more readily accomplished when the E1 primers are used in conjunction with E6 or E7 primers, as follows. For instance, one can amplify the E6/E7 region using the following E1 and E6/E7 primer pairs: (1) WD72, WD76 and TYN01, TYN02, TYN03; (2) WD64, WD 65 and TYN01, TYN02, TYN03; (3) WD72, WD76 and TYN04, TYN05, TYN06; (4) WD64, WD65 and TYN04, TYN05, TYN06; (5) WD72, WD76 and TYN07, TYN08; and (6) WD64, WD65 and TYN07, TYN08. In these latter amplifications, the entire E7 region is amplified. Thus, these amplification products can be detected with the E7 consensus probes depicted below:
__________________________________________________________________________TYP09 5' G A G C A A T T A G W A G A CTYP12 -- -- -- -- -- -- -- -- -- A R Y -- -- --__________________________________________________________________________
In another aspect of the invention an internal amplification control is used to assure that the competency of a sample for amplification and reduce the likelihood of false negative results. The internal control can be a genomic segment of DNA known to be present in a sample of human origin. For example, the .beta.-globin gene provides a suitable positive control amplification target. In one embodiment of the invention, .beta.-globin primers are included in the PCR reaction mixture for amplifying the L1 region of the HPV genome. .beta.-globin primers GH20 and PC04 produce a 268 base pair product.
(GH20 (Seq ID No. 83) 5'GAAGAGCCAAGGACAGGTAC
(PC04 (Seq ID No. 84) 5'CAACTTCATCCACGTTCACC
The .beta.-globin amplified segment is readily distinguished from the L1 amplified product by gel electrophoresis and by size (268 bp vs. 450 bp). For a positive control, when E6 consensus primers are used, other methods of detecting the .beta.-globin product are desirable because the E6 product is 240 base pair in size and, therefore, is not necessarily easily distinguished. In that event, a .beta.-globin probe can be used to detect the amplified product; PC03 is a suitable oligonucleotide probe.
PC03 (Seq ID No. 85) 5'ACACAACTGTGTTCACTAGC
Alternatively, the .beta.-globin and HPV amplification reactions can be run in parallel and analyzed individually. Similarly, it may be desirable to include positive and negative HPV controls in practicing the present methods for detecting and typing any HPV. For example, in one embodiment of the invention, plasmids containing genomic segments of HPVs 6, 11, 16, 18, and 33 are included. So long as the consensus primers, probes and type-specific probes used in the method can amplify and hybridize to the control nucleic acid, it is not essential that the control comprises the entire viral genome of each HPV. Example 2 describes specific HPV plasmids used as positive control; however, these specific plasmids are non-essential aspects of the invention and numerous cloned HPVs are described in the literature.
Those skilled in the art recognize that the specific primers and probes disclosed herein are merely illustrative of the invention. For instance, because the primers and many of the probes of the invention are single-stranded DNA molecules, and because the target DNA (HPV DNA in a sample) is double-stranded DNA, useful primers and probes of the invention can be constructed merely by synthesizing primers and probes complementary to those specifically disclosed herein. The primers and probes of the invention can also be prepared to amplify and detect sequence variations within areas of HPV genomes other than those specifically exemplified herein.
Primers of the invention are generally 18 to 21 nucleotides in length and are designed to have a high degree of homology with HPV sequences. For instance, in the design of the genital HPV consensus primers of the invention, a high degree of homology with all five major genital HPVs (HPV types 6, 11, 16, 18, and 33) was required. The consensus primers also are highly homologous to HPV31 (Goldsborough et al., 1989, Virology 171:306-311). For each region to be amplified, two regions of homology are required, one for negative-strand primers and another for positive-strand primers. To identify a homologous region, viral sequences are compared. Once a homologous region is identified, a consensus primer is designed. Degenerate bases can be used in the design to accommodate positions at which an individual virus varies in sequence from the homologous sequence. As many degenerate positions are made as is necessary so that all viral sequences have fewer than three mismatches with the consensus primer. The degenerate positions are chosen so that the smallest number of degenerate bases accommodates the largest possible number of vital sequences.
If a particular vital sequence has a large number of mismatches with the consensus sequence, then a type-specific primer is made for that virus. The type-specific primer is mixed with the degenerate primer that was designed for other viruses to make the consensus primer. Any mismatches that are not accommodated by the degenerate positions in the primer should generally be located more than 3 bases from the 3' end of the primer. Likewise, any degenerate positions should be more than 3 bases from the 3' end of the primer.
Estimated minimum and maximum T.sub.m s for a degenerate primer should be between 54 and 64 degrees C. T.sub.m s are estimated by the non-empirical formula: each G or C contributes 4 degrees C. to the T.sub.m ; each A or T contributes 2 degrees C. to the T.sub.m ; and the T.sub.m is the sum of the calculated values. Finally, primers should not be designed to span palindromes or repetitive sequences.
Short oligonucleotide consensus probes design is similar to consensus primer design, except that consensus probes generally do not contain as many mismatches as consensus primers. As a result, the T.sub.m for a probe can be higher than the T.sub.m for a primer. However, where a mismatch or degenerate position occurs with respect to the 3' end is not as critical for consensus probes as it is for consensus primers. Clearly, long probes can tolerate a greater number of mismatches because of the overall length of the probe.
Type-specific probes are designed so that a given probe will generally have less than 75% similarity with sequences from HPV types distinct from that recognized by the probe. The type-specific probes are usually 18-20 nucleotides in length with estimated T.sub.m s in the range of 58 to 64 degrees C. The present invention also describes that more than one type-specific probes may be employed for typing. This aspect of the invention provides additional assurance that variant types will be correctly detected and typed. Preferably two type specific probes are employed that are capable of hybridizing to opposite strands and residing in separate regions of the amplified product.
Those skilled in the art also recognize from the present disclosure that the method of the present invention can be carried out in a variety of ways. The present method is applicable to any human papillomavirus and especially preferred for detecting and typing genital HPVs. The method can be used to detect isolate-to-isolate variation within a particular HPV type and can also be used to screen for significant changes in the HPVs present in a patient. In one embodiment of the invention, consensus primers to more than one region of HPV DNA will be used, ensuring that if any portion of the HPV genome has been deleted, other regions can still be detected. In a similar fashion, the typing of the amplified DNA can be done using a variety of type-specific probes that recognize different regions of the amplified DNA. In a preferred embodiment, probes capable of hybridizing to at least two regions of the amplified DNA are employed to increase the likelihood of typing correctly HPV variants.
Those skilled in the an recognize that the present invention can also be used to detect HPV mRNA present in a sample. The expression of certain HPV mRNA species, particularly E6 and E7 mRNAs, may be indicative of the likelihood than an HPV infection will progress to carcinoma. To detect an HPV mRNA by the method of the present invention, the mRNA can be treated with reverse transcriptase in an appropriate reaction mixture to synthesize a cDNA molecule. The primer used in the reverse transcription reaction can be a consensus primer of the invention or can be a different oligonucleotide that hybridizes near the 3' end of the mRNA. Although random hexamers are not specific for the 3' end of the molecule, they are suitable for reverse transcription of RNA to provide a cDNA template for amplifying HPV nucleic acids. This cDNA copy is then made into a double stranded DNA molecule, which can be detected and typed in accordance with the method of the present invention.
The consensus primers of the present invention can also be used to detect HPV types previously uncharacterized. For instance, HPV isolate 88 noted in Table 4, above, has previously not been reported. Thus, the consensus primers of the invention can be used to amplify DNA sequences of previously unknown HPV types. The amplified DNA can then be sequenced and the sequence data used to generate type-specific probes for use in the method of the present invention.
The present invention may be assembled as a kit for detecting and typing HPV. Such a kit would include consensus primers and type-specific probes. A preferred kit also includes means for determining if amplification has occurred, such as a consensus probe or long probe. Additional kit components may include and are not limited to any of the following: PCR buffers and enzymes; positive control HPV or non-HPV DNAs; positive control primers, for example, .beta.-globin primers; a positive control probe; primers for preparing long probes; means for detecting hybridized probes; and instructions for amplifying, detecting, and typing HPV.
The examples provided below merely illustrate the invention and in no way limit the scope of the accompanying claims.
EXAMPLE 1
PCR-Based Detection and Typing of HPV
Preparation of Clinical Samples for PCR
The disclosed methods for detecting and typing HPV in a clinical sample are suitable for use with any sample that is suspected of containing HPV nucleic acids. It will be apparent to one of ordinary skill in the an that sample preparation procedures vary according to sample source. Specific examples are provided for sample preparation from a variety of sources.
A. Samples collected in ViraPap.TM. (Life Technologies Inc.) collection tubes were digested according to manufacturer's instructions. Samples were handled with extreme care to avoid cross-contamination (Kwok, 1989, Nature 339:237-238). Two hundred microliters of each sample was removed for PCR analysis with a disposable pipet, precipitated at -20.degree. C. overnight with 2M ammonium acetate and 70% ethanol, dried, and resuspended in 20 .mu.l TE (10 mM Tris-HCl, pH 7.5, 1mM EDTA). Two .mu.l and 5 .mu.l aliquotes were used for each amplification reaction. ViraPap.TM. collection tubes containing known amounts of purified human genomic DNA from the K562 cell line (ATCC #CCL243), which does not contain HPV DNA, were processed with each batch of 20 samples to monitor possible contamination in sample processing. The K562 cell line provided a negative control.
B. Freshly collected cells from clinical swabs were suspended in 1 ml of saline, pelleted by centrifugation, and the supernatant discarded. If cell suspensions were to be stored for a period of time before the test, antibiotics were generally added to the suspension (a commercially available antibiotic, such as Fungi Bact Solution, is suitable for this purpose). The cell number was estimated, and if blood was present in the sample, the cells were suspended in 1 ml of TE buffer to lyse the red blood cells and then repelleted. About 10.sup.2 -10.sup.4 cells were used for each PCR. The cells were first suspended in 100 .mu.l of buffer containing 50 mM Tris, pH 8.5; 1 mM EDTA; 1% Laureth-12; and 200 .mu.g/ml proteinase K. This mixture was incubated at 55 degrees C. for about 1 hour, and then the proteinase K was heat-inactivated by incubating the mixture at 95 degrees C. for 10 minutes. Aliquots containing 10.sup.2 -10.sup.4 cells were used per 100 .mu.l of mixture containing proteinase K treated cells.
C. Cervicovaginal lavage samples were stored frozen in 0.9% saline. For PCR the samples, as 1 ml aliquots, were thawed and mixed gently. Fifty microliters of sample was removed with a disposable piper and added to 50 .mu.l of 2X digestion solution in a labeled tube. The digestion solution was prepared fresh (1X digestion solution comprises 200 .mu.g/ml proteinase-K, 1% Laureth-12, 20 mM Tris-HCl pH 8.5, and 0.4 mM EDTA). The samples were incubated for one hour at 55.degree. C., spun briefly, and placed at 95.degree. C. for 10 minutes to heat inactivate the protease. The samples were again spun briefly and 5-10 .mu.l of each sample was added to a PCR mix for a 100 .mu.l PCR reaction.
D. The method of the present invention will often be used to determine whether tissue sections in paraffin contain HPV. To prepare such a tissue section, typically 5-10 .mu.M in width, for use in the present method, the following procedure was employed. The tissue section was extracted twice with 1 ml of octane to remove the paraffin. Each extraction was for about one-half hour. The tissue section was then rinsed twice with 100% ethanol to remove the extracting agent and dried. The section was then suspended in buffer with detergents and proteinase K and (200 .mu.g/ml proteinase-K, 1% Laureth-12, 50 mM Tris-HCl pH 8.5, and 1 mM EDTA) and incubated at 55.degree. C. for 2-4 hours. After heat inactivation of the proteinase K, the suspension was centrifuged to pellet debris, and about 1-20 .mu.l of the supernatant were used for each PCR reaction.
Control DNAs
HPV DNAs used in control amplifications were either recombinant plasmids containing HPVs (6, 11, 16, 18, 31, 33, 39, and 45) or DNA from cell lines containing HPVs (HPV16: SiHa [ATCC no. HTB35] or Caski [ATCC no.CRL 1550]; and HPV18: HeLa [ATCC no. CCL2]). DNA from the K562 cell line (ATCC no. CCL243) served as an HPV-negative control. Cellular DNA (3 to 15 ng) or plasmid DNA (1-10 pg) was used in control amplifications. Products from these amplifications were used to assess the specificity of type-specific probes.
PCR Protocols
All PCR protocols were carded out using a Perkin-Elmer/Cetus Instruments Thermal Cycler instrument. A typical reaction mixture contained 50 pmoles each L1 consensus primer MY09 and MY 11; 2.5 units of Taq polymerase; 10 .mu.l of 10X PCR buffer (0.5M KCl; 100 mM Tris, pH 8.5; 40 mM MgCl.sub.2); 0.2 mM of each dNTP; 1-20 .mu.l of each clinical, cell, or paraffin sample; and deionized water to 100 .mu.l. A 100 .mu.l mineral oil overlay was added to each reaction to avoid evaporation. When an amplification control was included in the reaction, 5 picomoles of each .beta.-globin primer (PC04 and GH20) (Seq ID Nos. 83 and 84) was added to the reaction mix.
Because of the extreme sensitivity of PCR, the potential exists for false positive results arising from sample cross-contamination or PCR product carry-over. Extreme care was taken to prevent contamination. Only disposable or positive displacement pipets were used in sample handling and PCR set-up. In addition, pre- and post-PCR samples and reagents were kept separated throughout all stages of the work. The extensive use of negative controls support the conclusion that contamination did not occur at any point in sample handling and all positive results represent true vital presence.
The clinical or cell sample aliquot was added immediately before temperature cycling. Tubes containing the K562 negative control DNA or no added DNA were included. PCR reaction times for use with clinical samples were as follows. The machine was heated to 85.degree. C. before the samples were placed into the machine. The machine was then programmed to execute the following temperature changes: 35 cycles of 60 seconds at 95.degree. C., 60 seconds at 55.degree. C. and 60 seconds at 72.degree. C.; 5 minutes at 72.degree. C.; and then hold at 15.degree. C.
For paraffin sections, the machine was programmed to reach 85.degree. C. and execute temperature changes as follows: forty cycles of 60 seconds at 95.degree. C., 60 seconds at 55.degree. C., and 2 minutes at 72.degree. C.; then 5 minutes at 72.degree. C.; and then hold at 15.degree. C. The MY09, MY 11 PCR product is less than 600 base pairs; however, if alternative primers were used and the PCR product was expected to be longer than 600 base pairs, the machine was programmed to execute temperature changes as follows: 1 minute at 85.degree. C.; forty cycles of a 50 second ramp to 95.degree. C., 50 seconds at 95.degree. C., a 50 second ramp to 55.degree. C., 50 seconds at 55.degree. C., a 50 second ramp to 72.degree. C., and 2 minutes at 72.degree. C.; 5 minutes at 72.degree. C.; and then hold at 15.degree. C.
Following amplification, 5 .mu.l of each completed reaction was electrophoresed on a 7% polyacrylamide gel, ethidium bromide stained, and photographed. Dot blots of each PCR amplification reaction were prepared.
Generic HPV Probe
A generic HPV probe was synthesized from 450 bp L1 PCR fragments of HPV16 (Caski), HPV18 (HeLa), PAP238B (a cloned clinical sample identified as HPV31 ) and a highly divergent, unidentified HPV sequence that was previously isolated and cloned from clinical specimens and is referred to here in as PAP88. PCR amplifications were performed separately using nested primers (Seq ID No. 1/Seq ID No. 2) MY74/MY75 for HPV16; MY76/MY77 (Seq ID No. 3/Seq ID No. 4) for HPV18; MY47/MY48 (Seq ID No. 5/Seq ID No. 6) for PAP88; and MY49/MY50 (Seq ID No. 7/Seq ID No. 8) for PAP238B. The L1 amplification products were used as the templates for preparing the generic probes. The buffer and enzyme conditions were used as described above except that the final 50 .mu.l PCR contained 50 .mu.M each dNTP, 62.5 pmol (50 .mu.Ci) each .alpha.-.sup.32 P-labeled dNTP, and 20 picomoles of each primer. The mineral oil overlay was omitted and the PCR cycling program was as follows: 30 seconds at 95.degree. C., 30 seconds at 55.degree. C., 1 minute at 72.degree. C. for 25 cycles with a final 5 minute extension at 72.degree. C. The PCR products from identical unlabeled reactions were visualized using electrophoresis and ethidium bromide staining. The labeled .about.400 bp fragments were purified using a G50 Sephadex column, and the cpm of the probes was determined using a scintillation counter. The four probes were denatured at 95.degree. C. for 10 minutes in the presence of sheared salmon sperm DNA, then rapidly cooled in a dry ice/ethanol bath before addition to the hybridization solution. Membranes were hybridized with a mixture of 1.0-1.5.times.10.sup.5 cpm/ml of each probe.
Hybridization Analysis of PCR Products
To determine if amplification had occurred the following protocol was used. About 2 .mu.l of each reaction mixture were added to 100 .mu.l of denaturing solution (0.4M NaOH and 25 mM EDTA) for each replicate dot and spotted onto replicate, positively-charged, nylon membranes (such as Genetran 45, Biodyne B Membrane [Pall], or Biotran from ICN) using a dot-blot or slot blot apparatus. The resulting dot was rinsed once with 200 .mu.l of 20XSS C. The membrane was then removed from the blotter, air-dried, and exposed to ultraviolet light (with the DNA facing the light) to covalently attach the DNA to the membrane (50 mjoules) by using a commercial UV crosslinking apparatus. Amplified L1 fragments from HPV types 6, 11, 16, 18, 31, 33, 35, 39, and 45 were spotted onto each membrane as positive controls to assess cross-hybridization and optimize exposure times.
The membrane was pre-hybridized at least 30 minutes at 65.degree. C. in a water bath or air incubator. The pre-hybridization solution contained 6XSSC, 5X Denhardt's solution, and 0.1% SDS and 100 .mu.g/ml denatured salmon sperm DNA. Alternatively, the membranes can merely be rinsed with pre-hybridization solution. .sup.32 P-labeled consensus probes (1.0-1.5.times.10.sup.5 cpm/ml of hybridization mix) were added to each filter and allowed to hybridize at 55.degree. C. for at 60-90 minutes. After the hybridization, the hybridization mix was decanted and the membrane was washed twice for 10 minutes in 2XSSC and 0.1% SDS at 56.degree. C. for the HPV generic and .beta.-globin probes. The membrane was then air-dried and allowed to expose X-ray film from 7 to 48 hours. Background was defined by negative controls which included amplified human genomic DNA.
To determine the HPV type of amplified DNA, PCR reaction mixtures were hybridized to type-specific probes as described above. The only significant difference in the procedure was that the final wash of the filter was done at a temperature very near the T.sub.m of the particular type-specific probe. Filters hybridized to different type-specific probes were not washed together. Specifically, membranes were hybridized individually with probes for HPV types 6/11, 16, 18, 31, 33, 39, 45, 51, 52, and 53, and PAP88, PAP155, and PAP251 as listed below. Type-specific oligonucleotide probes were .sup.32 P-labeled using a standard kinase reaction (Maniads et al. eds. Molecular Cloning, 1982, Cold Spring Harbor, N.Y.). Membranes were pre-hybridized and hybridized as described above, then washed as follows:
______________________________________Probe Target Wash Temperature______________________________________MY12 HPV6 57.degree. C.MY13 HPV11 57.degree. C.MY14 HPV16 59.degree. C.WD74 HPV18 59.degree. C.WD126 HPV31 57.degree. C.WD128 HPV31 57.degree. C.MY16 HPV33 57.degree. C.MY59 HPV33 57.degree. C.MY89 (SEQ ID NO. 32) HPV39 58.degree. C.MY90 (SEQ ID NO. 33) HPV39 58.degree. C.MY70 HPV45 59.degree. C.MY98 (SEQ ID NO. 43) HPV45 59.degree. C.MY87 (SEQ ID NO. 48) HPV51 58.degree. C.MY88 (SEQ ID NO. 49) HPV51 58.degree. C.MY81 (SEQ ID NO. 51) HPV52 57.degree. C.MY82 (SEQ ID NO. 52) HPV52 57.degree. C.MY101 (SEQ ID NO. 53) HPV53 58.degree. C.MY102 (SEQ ID NO. 54) HPV53 58.degree. C.MY103 (SEQ ID NO. 55) HPV53 58.degree. C.MY83 (SEQ ID NO. 68) PAP88 58.degree. C.MY84 (SEQ ID NO. 69) PAP88 58.degree. C.MY86 (SEQ ID NO. 70) PAP155 58.degree. C.MY93 (SEQ ID NO. 72) PAP251 58.degree. C.MY94 (SEQ ID NO. 73) PAP251 58.degree. C.______________________________________
Autoradiogram exposures that gave an intensity comparable to the generic probe were analyzed using the same standards described above. The sensitivity of this method was 100 copies of cloned HPV16 DNA or 10 HeLa cells containing 100-500 copies of HPV-18, per 1 ml collection tube when 2-5% was analyzed.
EXAMPLE 2
Dual Amplification of Archival Samples
A second set of consensus primers was designed to amplify a region of the E6 gene approximately 246 bp in length. These consensus primers were used in addition to the L1 consensus primers described in Example 1. The second set provides a means to generate corroborative HPV typing data and focuses on a gene which is consistently retained and expressed in tumors (Yee et al., 1985, Am. J. Pathol. 116:361-366; Seedoff et al., 1987, EMBO 6:139-144; and Broker et al., In Cancer Cells: Molecular Diagnostics of Human Cancer, vol. 7, Cold Spring Harbor, N.Y., 1989, pp. 197-208).
Archival tissue sections, which included 33 cases of cervical carcinoma, were subjected to PCR amplification with the L1 and E6 primer sets. The resulting products were analyzed by dot blot hybridization with consensus and type-specific oligonucleotide probes to illustrate the efficacy of the two site amplification strategy in the detection and typing of genital HPVs.
Tumor Samples
Sections from 48 paraffin-embedded tissue blocks representing 33 cases of cervical carcinoma and 7 HPV-negative control tissues were acquired from the pathology archives at the University of Amsterdam, the University of California at San Francisco, and the University of California at Davis. The case dates ranged from 1979 to 1988. Five micron sections were cut from tissue blocks with a microtome and the microtome blade was thoroughly cleaned with xylene between each specimen to minimize cross-contamination of samples. Dry sections were transferred to eppendorf tubes for PCR analysis and immediately adjacent sections were mounted and stained with hematoxylin and eosin for independent histological characterization. Preparation of DNA from tissue sections for PCR was done as described in Example 1.
HPV-negative tissues were interspersed with tumor samples during tissue preparation to detect any possible contamination during sample manipulation. The prepared samples were used immediately for PCR amplification, although storage at 20.degree. C. for up to two weeks resulted in only a nominal loss in specific amplification product yield. Control DNAs, described in Example 1, were included as specified below in the section "Dot Blot Hybridization of PCR Products."
PCR Amplification
L1 and E6 amplification reactions were performed using 1 and 10 .mu.l (0.5% and 5%) of each prepared sample. In addition to HPV-negative and HPV-positive control samples, "no DNA" controls were included during each amplification series to detect contamination during reaction set-up. Furthermore, only positive displacement pipets and disposable pipets were used in the assembly of amplification reactions to minimize possible contamination.
Each L1 amplification reaction contained 50 pmoles each of the L1 degenerate primers MY11 and MY09 as described (Example 1 ) and 5 pmoles each of the .beta.-globin primers GH20 (Seq ID No. 83) (5'GAAGAGCCAAGGACAGGTAC) and PC04 (Seq ID No. 84) (5'CAACTTCATCCACGTTCACC) was included for the simultaneous amplification of a human .beta.-globin product of 260 bp which served as an internal control.
The E6 reactions included 10 pmoles WD72 and 40 pmoles WD76 (the positive strand primers) and 10 pmoles WD66, 40 pmoles WD67, and 10 pmoles WD 154 (the negative strand primers) in a buffer containing 50 mM KCl, 10 mM Tris (8.3), 4 mM MgCl.sub.2, 200 .mu.M of each dNTP and 2.5 units of the recombinant AmpliTaq.RTM. polymerase (Perkin-Elmer Cetus Instruments).
Each reaction was subjected to forty amplification cycles in a DNA Thermal Cycler (Perkin Elmer Cetus Instruments) using thermocycle step parameters of 95.degree. C. for 1 minute, 55.degree. C. for 1 minute and 72.degree. C. for 2 minutes. An additional five minutes was included at the final 72.degree. C. elongation cycle. Thirty cycles were used in the amplification of HPV positive control DNA. Amplification products (1/20 of the reaction) were separated by electrophoresis using 7% polyacrylamide gels and visualized by UV illumination following ethidium bromide staining.
Probes
Oligonucleotide probes used for the dot blot hybridizations were labeled to a specific activity of 0.3-1X 10.sup.7 cpm/pmole with .alpha.-.sup.32 P ATP using T4 polynucleotide kinase (NENuclear/du Pont).
The consensus L1 probe was an equimolar mixture of the following four oligonucleotides corresponding to a conserved region located 50 base pairs (bp) from the 5'end of amplified region:
MY18 (5'CTGTTGTTGATACTACACGCAGTAC);
MY46 (Seq ID No. 9) (5'CTGTGGTAGATACCACWCGCAGTAC);
MY57 (5'CTGTGGTAGATACCACACGTAGTAC); and
WD 147 (5'CTGTAGTGGACACTACCCGCAGTAC).
L1 type-specific probes were MY12 for HPV6, MY13 for HPV11, MY14 for HPV16, WD74 for HPV18, WD126 and WD128 for HPV31, MY16 and MY59 for HPV33 and MY70 for HPV45. MY12 and MY13 were used together as a mixture to detect both HPV6 and 11. In clinical practice it is not necessary to separate HPV6 and HPV 11 type specific probes because these HPVs are highly related in sequence and in pathogenic potential. HPV6 and HPV11 are most often associated with benign lesions.
The use of two type specific probes for each HPV type provides additional assurance that all HPV DNAs will be typed correctly including variants, where nucleotide changes in the region of one type specific probe have occurred, will not be mistyped. Probes WD126, WD128, and WD170 (Seq ID No. 81) were designed using sequence information generated from amplification products of clinical isolates which were subsequently identified as HPV31 and HPV45. WD128 and WD126 reside in separate domains on the L1 product. WD128 detects both HPV31 and a variant referred to as HPV31b. The sequence of the HPV31b L1 product contains a restriction site not present in the archetypic HPV31. PC03 (Seq ID No. 85) (5'ACACAACTGTGTTCACTAGC) was used to identify the .beta.-globin product according to Example 1.
Oligonucleotide probes designed to distinguish E6 products were: WD 133 for HPV6, WD134 for HPV11, WD103 for HPV16, WD132 and WD170 (Seq ID No. 81) for HPV18, WD165 and WD166 for HPV31, RR1 and RR2 (Seq ID Nos. 79 and 82) for HPV33, and WD171 (Seq ID No. 80) for HPV45. The sequence of the HPV45 E6/E7 region was determined from pHPV45 kindly provided by K. Shah (Johns Hopkins University).
Dot Blot Hybridization of PCR Products
Replicate dot blots were prepared using 1/50 (2 .mu.l) of each amplification reaction. Additionally, control reactions (HPV positive SiHa DNA, HPV negative K562, and "no DNA") from each amplification series were included. Two .mu.l of each reaction were denatured in 100 .mu.l of 0.4N NaOH, 25 mM EDTA for each dot, applied to a Gentran 45 membrane (Hasco, Inc), and covalently bound by UV linking using a Stratalinker (Stratagene, Inc.) at 400 (X 100) .mu.Joules. The filters were washed in 0.1 X SSC, 0.5% SDS at 60.degree. C. for 30 minutes, followed by prehybridization for 15 minutes at 55.degree. C. with 6 X SSC, 5 X Denhardt's solution 0.5% SDS, 100 g/ml single-stranded sheared salmon sperm DNA.
Replicate filters were separately hybridized with .sup.32 P-labeled type-specific and generic oligonucleotide probes (1-200,000 cpm/ml) in 6 X SSC, 5 X Denhardt's solution 0.5% SDS, 100 .mu.g/ml single-stranded sheared salmon sperm DNA for 1 hour at 55.degree. C. Probes WD170 and WD171 (Seq ID Nos. 80 and 81) required hybridization at 45.degree. C.
Filters were rinsed briefly in 2 X SSC, 0.1% SDS at room temperature and then twice for 10 minutes at 45.degree. C. (WD170 and WD171) [Seq ID Nos. 80 and 81]); 50.degree.-52.degree. C. (WD132, RR1 and RR2[Seq ID Nos. 79 and 82]), 55.degree.-56.degree. C. (the consensus L1 probe mix, WD103, WD132, WD165, and WD166); 56.degree.-57.degree. C. (MY12/MY13, WD126, MY16, MY70, and WD133/WD134); or 58.degree.-59.degree. C. (MY14, WD128, MY59/MY64, MY70, and WD74). The membranes were subjected to autoradiography using Kodak XAR-5 film. L1 and E6 dot blot results were scored independently. The results indicated that for HPV45, MY69 may be a preferred type-specific probe, because MY70 has some sequence similarity to other HPVs.
Bands of predicted size for E6 or L1 were not detected in reactions from the normal cervical tissues nor an appendix. PCR products of the expected size for an L1 product were observed in all tumor samples with the exception of one of the older samples in the collection. Typing results with the E6 and L1 products were in complete agreement in all cased examined, except for one specimens which failed to produce an L1 PCR product. The single L1-negative specimen found in this study illustrates the value of a strategy based on two site analysis.
Other modifications of the embodiments of the invention described above that are obvious to those of ordinary skill in the areas of molecular biology, medical diagnostic technology, biochemistry, virology, genetics and related disciplines are intended to be within the scope of the accompanying claims.
EXAMPLE 3
Preparation of the Biotinylated Long Probes
In a non-isotopic detection format, Bio-11 (dUTP) (Sigma #B7645) was incorporated into the long L1 probe instead of .sup.32 P dNTP. Subsequent detection of the probe utilized a SA-HRP binding step and development of signal with Amersham's Enhanced ChemiLuminescence (ECL) system.
All reactions contained 2 .mu.l of a 1:100 or 1:1000 dilution of the appropriate MY09/MY11 generated L1 PCR product, 50 mM KCL, 10 mM Tris, pH 8.5, 2.5 units Taq polymerase, 200 .mu.M dATP, dCTP, dGTP, 100 .mu.M dTTP and 100 .mu.M Bio-11 dUTP. The primer and MgCl.sub.2 concentrations varied as follows: MY47 and MY48, 10 picomoles per primer and 4 mM MgCl.sub.2 ; MY49 and MY50(Seq ID Nos. 7 and 8), 10 picomoles each primer and 6 mM MgCl.sub.2 ; MY74 and MY75 (Seq ID Nos. 1 and 2), 10 picomoles each primer and 4 mM MgCl.sub.2 ; MY76 and MY77 (Seq ID Nos. 3 and 4), 50 picomoles each primer and 8 mM MgCl.sub.2. Each reaction was 200 .mu.l in volume with a 100 .mu.l mineral oil overlay. Thermal cycling parameters were 30 seconds at 95.degree. C., 30 seconds at 55.degree. C. and 1 minute at 72.degree. C. with a five minute extension at 72.degree. C. at the end of 30 cycles. The biotinylated PCR product was purified using a G50 Sephadex column (eluted with TE and 0.2% SDS) or an isopropanol, 2M ammonium acetate precipitation. Aliquots of both biotinylated and non-biotinylated long probes were electrophoresed on 7% acrylamide gels, ethidium bromide stained, and photographed under UV light. The non-biotinylated probe migrated further than the biotinylated probe.
The hybridization protocol using biotinylated probes was as described in Example I. Amersham's ECL system was used for detection according to the manufacture's instructions; however, the mount of SA-HRP used at the binding step was tinted for each lot of reagent.
Other modifications of the embodiments of the invention described above, that are obvious to those of ordinary skill in the areas of molecular biology, medical diagnostic technology, biochemistry, virology, genetics, and related disciplines, are intended to be within the scope of the invention and the accompanying claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 85(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CATTTGT TGGGGTAACCAAC20(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:TAGGT CTGCAGAAAACTTTTC21(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:TGT TTGCTGGCATAATCAAT20(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:T AAGTCTAAAGAAAACTTTTC21(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CATATGCTGGGGTAATCAGG20(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:CAGGTCTGCAGAAAAGCTGT20(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:TATTTGTTGGGGCAATCAG19(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CTAAATCTGCAGAAAACTTTT21(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 25 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:9:CTGTGGTAGATACCACWCGCAGTAC25(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)( xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:ACAATGAATCCYTCTGTTTTGG22(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:ATCGCCTCCMCCAAATG17(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:ACATACACCTCCAGCACCTA20(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CATACACCTCCAGCACCTA19(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GATATGGCAGCACATAATGAC21(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:AGTTTCTGAAGTAGATATGGCA22(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:CTGAAGTAGATATGGCAGCAC21(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(i i) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GTAACATCCCAGGCAATTG19(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GATGCTGCACCGGCTGAA18(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GCCCAGGTACAGGAGAC17(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:GGGCAATATGATGCTACCAAT21(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GTACCTGGGCAATATGATG19(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:TCTCCTGTACCTGGGCAA18(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D ) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:TCAGCCGGTGCAGCATCC18(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GAGGGAGGTGTGGTCAAT18(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:AAGAACCTGAGGGAGGT17(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:CCAAAAGCCYAAGGAAGATC20(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:ACCACACCTCCCTCAG16(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:ACAGGCCATTACATGTCAA19(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:CTGCTGTGTCTTCTAGTGACAG22(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:TGCACCAAAACCTAAAGATG20(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:ATCATCTTTAGGTTTTGGTGC21(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid( C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:TAGAGTCTTCCATACCTTCTAC22(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:CTGTAGCTCCTCCACCATCT20(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:AGACACTTACAGATACCTACAG22(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:CACACCAGGCCCATATAAT19(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:CCAAGGTACGGGAGGATC18(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:GATCCTCCCGTACCTTG17(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:CACTGCAACATCTGGTGAT19(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:TCACCAGATGTTGCAGTG18(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:GGCGTTGTTACCTTAGCC18(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs( B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:GGCTAAGGTAACAACGCC18(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:GGATACTACACCTCCAG17(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:GCACAGGATTTTGTGTAGAGG21(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:TGTATTTGGCACAGGATTTTG21(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:CAGGATTTTGTGTAGAGGCA20(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:CAAATCCTGTGCCAGGTAC19(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:GCACAGGATTTTGTGTAGAG20(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:TATTAGCACTGCCACTGCTG20(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:CCCAACATTTACTCCAAGTAAC22(2) INFORMATION FOR SEQ ID NO:50:(i) SEQUENCE CHARACTERISTICS:(A ) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:CTGAGGTTAGAAAGGAAAGCA21(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:CACTTCTACTGCTATAACTTGT22(2) INFORMATION FOR SEQ ID NO:52:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:ACACACCACCTAAAGGAAAGG21(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:CGCAACCACACAGTCTATGT20(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:TTCTACCTTACTGGAAGACTGG22(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:GGAGGTCAATTTGCAAAAC19(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:TGCAGGGGCATTATTCTTT19(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:TACAGCATCCACGCAG16(2) INFORMATION FOR SEQ ID NO:58:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:CACGCAGGATAGCTT15(2) INFORMATION FOR SEQ ID NO:59:( i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:CCACGCAGGATAGCTT16(2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:GTGCTGCTACAACTCAGTCT20(2) INFORMATION FOR SEQ ID NO:61: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:GCTACAACTCAGTCTCCATC20(2) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:TGCCTTTTCAGGGGGAG17(2) INFORMATION FOR SEQ ID NO:63:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:AATGTCTCTTTGTGTGCCAC20(2) INFORMATION FOR SEQ ID NO:64:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:GTGTGCCACTGTAACCACA19(2) INFORMATION FOR SEQ ID NO:65:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:GGATCAGTAGGGGTCTTAGG20(2) INFORMATION FOR SEQ ID NO:66:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:GCCAGTTAAACAGGACCC18( 2) INFORMATION FOR SEQ ID NO:67:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:CATAAGGGTCCTGTTTAACTG21 (2) INFORMATION FOR SEQ ID NO:68:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:ATTAATGCAGCTAAAAGCACATT23(2) INFORMATION FOR SEQ ID NO:69:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:GATGCCCGTGAAATCAATCAA2 1(2) INFORMATION FOR SEQ ID NO:70:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:TACTTGCAGTCTCGCGCCA 19(2) INFORMATION FOR SEQ ID NO:71:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:CCAACACCGAATCAGAATATAAA 23(2) INFORMATION FOR SEQ ID NO:72:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:GCACTGAAGTAACTAAGGAAGG 22(2) INFORMATION FOR SEQ ID NO:73:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:AGCACCCCCTAAAGAAAAGGA 21(2) INFORMATION FOR SEQ ID NO:74:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:GTAGGTACACAGGCTAGTAGCTC 23(2) INFORMATION FOR SEQ ID NO:75:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:GCTCTACTACAACGTATGCCA 21(2) INFORMATION FOR SEQ ID NO:76:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:AGTTGCCAACGTCCTCAAC 19(2) INFORMATION FOR SEQ ID NO:77:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:GAGATWTATKCATATGC 17(2) INFORMATION FOR SEQ ID NO:78:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:GAGGTATWTGAHTTTGC 17(2) INFORMATION FOR SEQ ID NO:79:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:GTACTGCACGACTATGT 17(2) INFORMATION FOR SEQ ID NO:80:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:ACAAGACGTATCTATTG 17(2) INFORMATION FOR SEQ ID NO:81:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:GCAAGACATAGAAATAA 17(2) INFORMATION FOR SEQ ID NO:82:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:ACCTTTGCAACGATCTG 17(2) INFORMATION FOR SEQ ID NO:83:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:GAAGAGCCAAGGACAGGTAC 20(2) INFORMATION FOR SEQ ID NO:84:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:CAACTTCATCCACGTTCACC 20(2) INFORMATION FOR SEQ ID NO:85:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:ACACAACTGTGTTCACTAGC 20

Number	Name	Date
4849331	Lorinez	Jul 1989
4849332	Lorinez	Jul 1989
4849334	Lorinez	Jul 1989
4908306	Lorincz	Mar 1990
4983728	Herzog et al.	Jan 1991
5057411	Lancaster et al.	Oct 1991

Number	Date	Country
0354440	Jan 1989	EPX
0425995	May 1991	EPX
8605816	Oct 1986	WOX
8806634	Sep 1988	WOX
8902934	Apr 1989	WOX
8909940	Oct 1989	WOX
9108313	Jun 1991	WOX

	Number	Date
Parent	3747	Sep 1989
Parent	322550	Mar 1989
Parent	243486	Sep 1988

Detection of human papillomavirus by the polymerase chain reaction

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CPC

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Abstract

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US Referenced Citations (6)

Foreign Referenced Citations (7)

Non-Patent Literature Citations (61)

Continuations (1)

Continuation in Parts (3)

Entry
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