DETECTION OF HPV TRANSCRIPTS

Abstract: A method for detecting and/or quantitating specific DNA and/or RNA transcripts of human papillomavirus (HPV) is described. Nucleic acid probes specific for the DNA or the transcripts are used to determine the amount of the DNA or transcripts, and to ascertain the degree of amplification of certain high-oncogenic HPV genes. The method provides an accurate and reliable method for prognosticating serious cervical neoplasias and cancers.

Description: DETECT1.0N_0F_HPV_TRANS ^ CRI.PTS __!_ £__iE-_------ Α £.l£.&I.2.}-\Ω. . Epidemiologic data are accumulating which show that the human papillomavirus (HPV) is strongly associated, as a factor or co-factor, with cervical cytologic abnormalities, such as cervical intraepithelial neoplasias (CIN) and carcinoma in i.tu (CIS). Syrjanen, K.J. , K. Syrjanen, L. Gissman, and L.G. Koss (Eds.), Springer-Verlag, pp 467-503, (1987) . In general, these dysplasias are found in the transition zone of the cervix and are graded from mild to severe (I to III), based on the extent to which neoplastic cells extend from the basal layer to the epithelial surface. Complete replacement of the epithelium by neoplastic cells is termed carcinoma i ^ n sjLtu (CIS). At least 60 types of HPV, isolated from various parts of the human body, have been documented, and more than 20 of these types have been shown to be associated with the genital mucosa. E. M. de Villiers, J^Virol^, (53£11 .:4898-4903 (1989). Although present information strongly supports the close association between HPV and cervical neoplasia, and shows that many individuals clearly have HPV DNA (i.e. , infections), transiently and/or latently, it also makes it clear that women in whom HPV is present in cervical cells do not always progress to more serious dysplasia. Syrjanen et al . in Cancer_Cells, Vol. 5, Cold Spring Harbor, pp. 281-288 (1987); deBrux et_a1.. , Bu11^_Cancer_£Pari. 2, 20:410-422 (1983); Mitchell et_al . , The_Lancet , 1:573-575 (1986) . Thus, simple detection of HPV DNA in cervical specimens lacks discriminatory predictive value for identification of women at risk for serious disease. A method for assessing HPV activity which can be used to predict progression to serious CIN or CIS is needed. The present invention relates to a method for detecting and quantitating specific transcripts of HPV and/or HPV DNA in cervical specimens and, in addition, to a method of determining patterns of HPV transcription which are more reliable indicators or predictors of the risk of progression to serious dysplasia or cervical carcinoma in individuals in whom HPV is present than is measurement of total HPV DNA in a sample. The method involves detecting and/or quantitating RNA, DNA or RNA/DNA ratios which are indicative of the presence or absence of high- oncogenic types of HPV. For example, one embodiment of the method involves using nucleic acid probes and/or probe sets comprising DNA or RNA sequences which hybridize to transcripts of the E6 and/or E7 genes of specific HPV types, particularly to HPV types associated with oncogenesis, referred to as high-oncogenic HPV types, (e.g. , HPV 16, 18, 31, 33, 35, 39, 45, 51, 52 and 56), but not to low-oncogenic HPV types (e.g. , HPV 6 and 11). The present method can also be used to detect and/or quantitate low-oncogenic HPV types (e.g. , HPV 6 and HPV 11), if desired (e.g. , as an indication of lower risk of progression to serious CIN or CIS in an individual having an HPV infection than would be the case if high-oncogenic HPV types are present) . The present method is based on hybridization of selected nucleic acid probes for detection and/or quantitation of specific HPV messenger RNAs (mRNAs) or DNA; thus it provides a means for assessing active transcription of the HPV genome. Type-specific nucleic acid probes and probe sets for HPV are also the subject of the present invention. These probes are of two types: poly- nucleotide -tailed oligonucleotide capture probes and labelled detector probes (also referred to as labelled riboprobes). Tailed capture probes serve two purposes: 1) they hybridize to at least a portion of an open reading frame (ORF) sequence of an HPV gene or to at least a portion of the encoded transcript of a selected HPV type which is associ¬ ated with ^normalities, progression to higher grade CIN or development of CIS in cervical epithelial cells; and 2) they link (through the polynucleotide tail) the hybridization complex formed as a result of the method described to a solid support, thus making it possible to separate the hybridization complex from the remainder of the sample. Labelled detector probes are generally single-stranded RNA molecules or single stranded DNA molecules which hybridize to portions of bi- or polycistronic transcripts of genes associated with cervical cytologic abnormalities, such as the E6 and/or E7 genes, or to spliced transcripts which contain these ORF's or portions thereof, of selected HPV types; specifically, labelled detector probes form stable hybrids with these transcripts, including spliced transcripts. Although it is possible to use detec¬ tor probes which hybridize to target sequences which also are recognized by capture probes, it is prefer¬ able that they do not. In addition, probes for specific detection of spliced transcripts of the E6 gene of HPV 16, HPV 18, HPV 31, HPV 33 and HPV 35 are the subject of the present invention. Such probes are splice-junction capture probes designed to form stable hybrids only with spliced transcripts of a selected HPV type and they serve as examples of a class of probes which are useful in the present method. A method for determining prognosis in in¬ dividuals infected with HPV is also the subject of the present invention. The method is based on the association between HPV transcriptional activity and the risk for development of serious cervical dys- plasia or carcinoma. For example, patterns and/or levels of transcription of at least a portion of the E6 and/or E7 genes and spliced downstream sequences of selected high-oncogenic HPV types can be used to predict the progression of HPV infection to serious CIN or CIS. Conversely, patterns and/or levels of transcription of the E6 or E7 genes of low-oncogenic HPV types can be used as an indication of reduced risk of serious CIN or CIS. Thus, the detection of E6 and/or E7 transcripts of selected HPV types in cervical samples provides a more valuable diagnostic aid than mere detection of HPV DNA. Certain patterns of HPV transcription can provide a more reliable indicator of risk for progression to serious dysplasia than does detection of total HPV DNA. Low levels of HPV DNA are quite prevalent in women and, thus, the presence of HPV DNA is not a sensitive measure of neoplastic risk. The detection and quantitation of transcripts of genes associated with high-oncogenic or low- oncogenic types of HPV, and/or spliced mRNAs con¬ taining these sequences provide a sensitive, ac¬ curate, reliable prognosticator of serious dysplasia or cancer. Brie_f_D £ri£ i£n_of_ he__Fi^ure> Figure 1 is a schematic representation of the method of the present invention. Figure 2 is a schematic illustration of the hybridization complex of mRNA targets, d(A) -tailed oligonucleotide capture probes, and radiolabeled detector probes for detection of E6/E7-containing transcripts of HPV. Figure 3 shows the nucleotide sequences of thirty-three type-specific oligonucleotide probes, which range in length from 32 to 42 nucleotides. Figure 4 shows nucleotide sequences and target sites of probes for type-specific detection of spliced E6 transcripts (E6*)of HPV 16, HPV 18, HPV 31 and HPV 38. Predicted base pairs between spliced E6* transcripts and specific oligonucleotide probes which span the upstream and downstream exons are shown. Figure 5 is a schematic illustration of capture and detector probes on E6/E7 transcripts of HPV 16, showing the specific arrangement of probes on RNAs of HPV 16. Splice donor (do) and acceptor (ac) sites in the E6 ORF are indicated. Figure 6 shows nucleotide sequences of possible base pairs between HPV 16 capture probes 16-3, 16-4, and 16-5 and aligned sequences in the E7 ORF' s of HPV 6, HPV 11, HPV 18, HPV 31, HPV 33 and HPV 35. Underlined bases are predicted to form base pairs with the oligonucleotide probe. Figure 7 is a schematic illustration of the steps for cloning HPV 16 DNA into lambda 47.1 and construction of recombinant molecules for synthesis of detector probes and positive target transcripts. The strategy for engineering a construct for production of the HPV 16 riboprobe is shown on the left side of the figure and the construction of a recombinant molecule for synthesis i.n vitro of mRNA molecules which contain the E6 and E7 ORF's (i.e. , positive target transcripts) is shown on the right side of the figure. Figure 8 shows the relationship between the concentration of positive target and signal in an HPV 16 assay. Figure 8A shows typical results from 32 the HPV 16 assay using a P-labeled detector probe o with a specific activity of 5 x 10 cpm/μg. Figure 8B is a graph showing the linear relationship between the concentration of positive target and the amount of signal. D£_& ii£iϊ_£ £_!i._£-ti££_£f_.E^ The present invention is based on an under¬ standing of the close association between HPV and cervical carcinoma; the fact that although many individuals have HPV DNA in cervical cells, they do not progress to more serious dysplasia; and a deter¬ mination that simple detection of HPV DNA in cervi¬ cal specimens appears to lack discriminatory pre¬ dictive value for identification of individuals at risk for progression to serious disease. In contrast to commonly used techniques for detection of HPV DNA, the present method allows sensitive and quantitative detection of specific HPV mRNA or DNA. The present method makes it possible to assess active transcription of the HPV genome and to determine the associations between HPV transcrip¬ tion patterns and risk for development of cyto- pathology and/or progresson to more serious disease (i.e. , higher grade CIN or CIS) in HPV-infected individuals. The following is a brief summary of the assessment of the role of HPV in cervical abnormalities, which establishes the need, and therefore the utility of the present method, a detailed description of the method, a description of HPV-specific nucleic acid probes useful in the method, and discussion of an application of the method in a clinical context for diagnostic and/or therapy-related purposes. Th£_R£l ^ e_£f_HPV_^n_ ervi£a ] ._Abn£rmal.^ti.e The role of HPV in cervical abnormalities, which range in severity from mild dysplasia to complete replacement of the cervical epithelium (CIS), has been studied extensively. A positive correlation between grade of dys¬ plasia (i.e. , CIN I, II or III and CIS) and the presence of HPV has been established. Kurman et a1. , Am_^_J ._0b tet^Gy.ne£ l^, 15 :293-296 (1988); Koutsky et al . , E£idem^_Rev^, 10:122-163 (1988); deVilliers et al . , The_Lan£et, 2:703-706 (1987) . It has been determined that higher grade cytologic abnormalities are more often associated with HPV types 16, 18, 31 and 33 than with low-oncogenic HPV types. HPV types 6, 11, 16 and 18 are found in 80-90% of dysplasias. Koutsky et al . , ibid. However, HPV DNA also has been shown to be present in cervical samples evidencing no ab¬ normality. For example, assays of cervical speci¬ mens for HPV DNA of types 6, 11, 16 and 18 by polymerase chain reaction (PCR) from 150 women with no cytologic abnormalities revealed that HPV DNA was present in 70-84% of the women. Young et al. , B£itish_Med^_J^, 298£66652:11-14 (1989); Tidy et al. , The_Lancet, (Feb. 25, 1989) p. 434. Relatively long-term epidemiologic studies which assess clinical progression from HPV-associ- ated infections (koilocytosis and/or HPV DNA) to CIN I or greater suggest that 8-15% of HPV infections became more dysplastic with time. Syrjanen, K. et al. , Zϋ;£ il£S£ZirH £ l_Can£er_Cell£, V£l.^__5_, Cold Spring Harbor, pp 281-288, (1987); de Brux et al. , , 20:410-422, (1983); Mitchell et al. , The_Lanc£t, 1:573-575, (1986); Syrjanen, K. et a1. , J__Ce1luiar_Bi£ h£2__.._._.__HE£I• » i ≤» P • - 98 (1989) . Conversely, this suggests that 85-92% of HPV infections do not progress with time. Progres¬ sion was observed more frequently (about 35%) in in¬ dividuals infected with HPV 16 than with types 6, 11, 18, 31, and 33 (7.5%-20%). It is unclear, at least, and unlikely, at best, that the presence of HPV DNA alone is a prognostic indicator of progres¬ sion to serious dysplasias, at least in part because co-factors very likely play roles in disease progression. Several lines of evidence suggest that expres¬ sion of the E6 and/or E7 genes of HPV is necessary for oncogenesis. The expression of these genes causes transformation of rat or mouse epithelial or fibroblast cells (Crook et al. , P o£_ : _Na ^ ._ : A ad^ J_£i._.. .85:8820-8824 (1988); Watanabe and Yoshiike, l£ LI -_!__._£££££r , 41:896-900 (1988)), and, after insertion into a retroviral vector, produces tumors in nude mice. Yutsudo et al . , " Vir^i^, 1.6_6_: 594- 597 (1988). Phelps and co-workers showed that the E7 gene can cooperate with an activated r_a.s oncogene to transform primary baby rat kidney cells and can transactivate a heterologous viral promoter. They also showed that portions of the predicted E7 protein are similar to several of the conserved domains of the adenovirus Ela protein, a trans- activating protein which can also cooperate with an activated ras oncogene to transform rat epithelial cells. Furthermore, continued expression of the E7 gene seems to be required for maintenance of the transformed phenotype in cells which have been transformed by HPV 16 and EJ-ra Banks et al . , J^ Ce11u1ar_Bioche . , £u££l^_13.C : 202 (1988); Crook et al. , EMB0_j£urna1 , .£.22: 513-519 (1989). The E7 protein is localized in the cytoplasm and has been shown, by Western blot analysis, to be the most abundant HPV protein in cell lines containing HPV 16 DNA (CaSki and SiHa) or HPV 18 (HeLa, C4-1, and SW756). Seedorf et al . , EMB0_J^, 6. 2 * 139-144 (1987) . In all HPV-containing cervical carcinomas, and cell lines derived from them, the E6 and E7 genes are intact. Wilzynski et al . , aroi^, J.6_6_: 624-627 (1988). Furthermore, transcripts of the E6 and/or E7 gene, but not necessarily other early genes, are consistently found in carcinomas and carcinoma cell lines. Baker et al . , J.-._Vir£!_;_, 6 .4.: 962-971 (1987); Pater and Pater, Can£er_Res^, 4^:324-328 (1988); Schneider-Gadicke and Schwarz, EMB0_Journa1 , 5^2 : 2285 -2292 (1986); Schwarz et al . , Inj. Eili£5 ∑ ££ £ _££d_Human_Di ea e • Springer-Verlag, pp. 443-466 (1987); Shirasawa et al . , J^Gen^ V r£l_^, 6_ :583-591 (1987); Smotkin and Wettstein, _-£ ___-i £li_A£≤ ; i^___i i^. 13:4680-4684 (1986) . The evidence suggests that expression of the E6 and/or E7 genes of HPV 16 and 18 are important for cellular transformation and may be important to the development of CIS. Thus, although presently-available data suggest a strong association between HPV DNA in cervical samples and cervical abnormalities which range in severity from mild dysplasia to CIS, they also -make it clear that many, and perhaps most, women in whom HPV DNA is present in cervical samples do not progress to dysplasia. Patterns of transcription, including spliced transcripts, have been examined for several HPV types with high oncogenic potential. Examination of three cell lines containing HPV 18 has shown three patterns of transcription (ascertained by analysis of cDNA clones). Schwarz et al . , Nature > 314:111-114 (1985) . Pattern 1 contains full length transcripts of the E6 and E7 genes and the 5' -terminal 11 nucleotides (nts) of the El RNA (El starts at nt 914; splice donor site at nt 925) spliced to 3' cellular sequences. Patterns 2 and 3 contain shortened transcripts of E6 (intron of 182 nts missing between a splice donor site at nts 226-234 and the splice acceptor site at nts 401-412), designated E6*, which are joined to sequences of E7 and either downstream cellular sequences (Pattern 2) or to downstream HPV sequences. (Pattern 3). E6* splice sites also are present in DNA sequences of HPV 16, HPV 31, HPV 33, HPV 35, HPV 43, HPV 52 and HPV 56 but not in DNA of low-oncogenic HPV types (i.e. , 6 and 11) Goldsborough et al . , Vir£l . , 121:306-311 (1989). Therefore, the E6* transcripts may be related to oncogenic potential of the HPV types. E6/E6* and E7 transcripts are found in HPV 16-containing cells, but they also contain spliced downstream E2-E4 exons , followed by cellular sequences. Two HPV 16 E6* transcripts have been identified in SiHa cells and two cancers. Smotkin et al , J_. ir£l_^ .lill '- 1441-1447 (1989). Both of them use a common donor splice site at nts 224-232 and a splice acceptor site at either nts 399-417 (E6*I) or at nt 516-543 (E6*II). Because translation of E6*I terminates at a greater distance from the E7 translation start signal (AUG) than does translation termination of E6*II, the former transcript may allow more efficient translation of the E7 ORF. Such transcription patterns described above form the basis for the design of probes and for their use in the method of detection and/or quanti- tation of specific transcripts of HPV in samples in the present invention. In addition, they provide the basis by which the present method is used for assessing risk of progression of cerival abnormali¬ ties. They provide the basis by which the present method is employed to detect and/or quantitate the above-described transcription patterns and/or other transcription patterns for assessing risk of pro¬ gression of cervical abnormalities to more serious disease. As will be explained and illustrated, the present method is one in which RNA, DNA or the ratio of RNA to DNA of specific HPV types in cervical samples are detected and/or quantitated as a measure of neoplastic risk. Detection and/or quantitation of E6 , E7 and spliced transripts of HPV types associated with oncogenesis or progression of cervical dysplasias as a means of determining neo¬ plastic risk provides a novel tool which can be used not only in a diagnostic context (i.e., to assess presence or absence of risk of progression) , but also in a therapeutic context (i.e. , as a means by which treatment can be designed and/or monitored) . D£tecti£n_of_S2ecif c_HPV_Transcri£t _bχ_the_Present Method The present method specifically involves detection and/or quantitation of RNA of selected HPV types which are associated with oncogenesis. In particular, the expression of the E6 and/or E7 genes, which can include downstream genes, and their expression relative to other genes is believed to be a prerequisite for HPV-related oncogenesis. In one embodiment of the present method, HPV transcription levels, particularly transcripts of at least a portion of the E6 and/or E7 genes of selected HPV types, are measured. That is, the amount of E6 and/or E7 mRNA present in a sample is detected and quantitated. Bicistronic and/or spliced transcripts of these genes also can be measured. ϊ_l£_M i_l _ϊ__£ _£_l _∑2_. _E. £j_i£Σ £_r £2 The present invention is a nucleic acid hybridization method which makes use of two types of nucleic acid probes, each of which is complementary in sequence to and capable of hybridizing to at least a region of the RNA transcript of the gene of at least one HPV type associated with oncogenesis or to an HPV gene. In particular, HPV DNA or HPV mRNA transcripts expressing at least a portion of the E6 and/or E7 genes, are detected and/or quantitated using the method. The method is represented schematically in Figure 1 and is described below as it can be applied to any selected HPV type. It is further described, with reference to a specific HPV type (HPV 16) . The sample to be assayed by the present method for E6/E7 and other spliced mRNA transcripts will generally be, for example, a cervical scrape, smear, mucus specimen, biopsy or tumor specimen. The sample does not have to be separated, filtered or precultured prior to being assayed by the present method. The sample can be mixed or diluted in a medium, if appropriate, and, if necessary, pretreated with an agent which disrupts cell and molecular structures within the cells. This disruption step frees the nucleic acids to be detected (the target nucleic acids). Cells can be disrupted, for example, using chaotropic agents which disrupt the molecular structure of a cell. That is, the agent denatures the secondary, tertiary and/or quaternary structures of biopolymers, including proteins, nucleic acids and polysacharides , which are generally found in biological specimens. Examples of chaotropic agents include chaotropic salts (e.g. , guanidiniu thiocyanate) , hydrolytic enzymes (e.g. , proteases, RNAases) and compounds that disrupt hydrophobic interactions (e.g. , sodium dodecyl sulfate, phenols, dimethylformamide , dimethyl sulfoxide, tetramethyl urea or guanidinium hydrochloride) . Physical or mechanical means of disrupting molecular structures, e.g. , sonication, can also be used to release nucleic acids. If necessary, nucleic acids present in the cells and released from the cells are also treated to render them available for hybridization with complementary nucleic acid sequences (e.g. , by heating to render double stranded sequences single stranded) . Two types of HPV type-specific probes are then contacted with the sample, either simultaneously or sequentially. One is the HPV type-specific tailed capture probes, which can be free or bound to a solid support. The capture probes are allowed to hybridize with the target transcripts in the sample mixture, forming a capture probe/target transcript complex. Either along with the capture probe or after the capture probe/ target transcript complex is formed, labeled detector probes are also contacted with the sample, which is maintained under appropriate conditions (e.g. , time, temperature, pH, salt concentration, chaotrope concentration) for hybridization of complementary nucleotide sequences to occur and form a capture probe/target transcript/ detector probe complex. The capture probe/target transcript/detector probe complex, which, for convenience, is subsequently referred to as the hybridization complex, is recovered from the sample mixture. This recovery is carried out by adding to the mixture containing hybridization complexes a solid substrate which is coated with a polynucleotide complementary to the tail of the capture probe. The tail of the capture probe present in the hybridization complex hybridizes with the complementary substrate, making it possible to separate the entire hybridization complex from the sample. In the case of pre-hybridized capture probes, the substrate containing the attached probe is removed from the sample mixture. Intact hybridi¬ zation complexes may be released from the solid substrate by treatments which reversibly disrupt base pairings (the capture probe forms fewer base pairs with the solid substrate than do the probes with the target transcript) and complexes may be recaptured on to other solid substrates; the result of this cycling of hybridization complexes is reduction of noise which usually binds non-specifi- cally to the solid substrate. The quantity of detector probes and, thus, of the specific HPV transcript, present on the separated substrate can then be determined using known techniques, such as scintillation counting, densitometric scans of autoradiographs, fluorescence spectroscopy, and beta emission counters (e.g. , Betagen) . The method used will depend upon the detector probe label. The amount of signal is related to the amount of target nucleic acids cap¬ tured, which in turn is proportional to the amount of transcript in the sample. The amount of transcript is related to the level of transcrip¬ tional activity of the selected gene (e.g. , E6/E7 genes), which activity is indicative of the neo¬ plastic risk. Thus, the method provides a sensitive assay for determining the risk of progression of HPV infection to serious CIN or CIS. In the embodiment of the present Invention in which the occurrence (presence or absence) of selected HPV types is determined or quantitated in order to assess risk of progression to serious cervical dysplasia or cancer in sLtu, the procedure is as follows: as described above, the presence or absence and/or quantity of one or more selected HPV types is determined. These types can be high- oncogenic HPV types, low-oncogenic types or both. The results of this assessment are compared with a previously-established relationship between the HPV types detected or quantitated and risk of progres¬ sion to serious dysplasia of the cervix or to cervical carcinoma. Such a relationship can be determined by using the previously-described method of detecting and/or quantitating selected HPV types in women whose clinical characteristics (e.g. , extent of progression of the condition) is also assessed and related to patterns of HPV DNA present and/or patterns of transcription. S-?Y-L££ecific_Nu£lei£_Acid_Pr£bes Nucleic acid probes and probe sets which are specific for HPV genes associated with cervical cytologic abnormalities which often progress to more serious CIN or CIS, are used in the present method. Probes which are particularly useful in the present method are oligonucleotide probes having a nucleo¬ tide sequence which is complementary to at least a portion of the E6 and/or E7 ORF, or spliced portions thereof. A nucleotide sequence is complementary to a second or target nucleotide sequence (e.g. , the E6 or E7 gene of HPV) if it hybridizes and remains hybridized to the second or target nucleotide sequence under the conditions used in the method. Two types of probes are used: tailed oligo¬ nucleotide "capture probes" and labelled "detector probes" (also referred to as labelled riboprobes). Tailed capture probes serve two purposes: they are complementary to/ hybridize to at least a portion of the ORF sequence of a gene of a selected HPV type or of the encoded transcript (mRNA) and they link the hybridization complex (capture probe/target nucleo¬ tide sequence/detector probe) to a solid support, thus making it possible to separate the hybridiza¬ tion complex from the remainder of the sample. A schematic representation of the complex of detector probe, target transcript, capture probes and solid support is shown in Figure 2. Labelled detector probes are generally single-stranded RNA or DNA probes which hybridize to portions of the bi- or polycistronic transcripts of genes of selected selected HPV types; specifically, they hybridize to portions of these genes which can be, but preferably are not also recognized by the capture probes. The selected HPV types are either high-oncogenic or low-oncogenic . The capture probes are characterized by a poly- nucleotide tail which is generally formed from a nucleotide homopolymer, such as polydeoxyribo- adenylate (poly(dA)), polydeoxyribocytidylate (ρoly(dC)) , polydeoxyriboguanylate (poly (dG)), and polydeoxyribothymidylate (poly(dT)). The probe tail is complementary to a polynucleotide sequence which is affixed to a solid support, such as magnetic beads, polysyrene beads and polystyrene dipsticks, allowing the probe to be captured and separated from the test sample. Probes particularly useful in the present Invention as capture probes have the following characteristics : 1. They are synthesized easily by n v tro transcription (for detector probe) or by chemical means (for oligonucleotide capture probes). Detector probes should be easily labeled, and to high specific activities, during in y .tr£ transcription or chemical synthesis or by post-synthetic modification with either isotopic or non-isotopic markers which can be easily quantitated. 2. They are HPV type-specific . Although HPV 6 sometimes has been associated with carcinomas, it and HPV 11 most often are associated with low risk for progression to high grade dyspla¬ sia. Because E6 and E7 genes of HPV 6 and HPV 11 show high sequence conservation, probes to these regions may cross -hybridize ; this result would not detract from the efficacy of the assay. Probes which are particularly useful in the present assays are those specific for HPV 16, 18, 31, 33 and 35. 3. They are of sufficient length and nucleotide sequence that they do not dissociate from specific target sequences on viral transcripts in the assay format, and hybridize specifically and quantitatively to transcripts of HPV. 4. They hybridize to selected HPV DNA sequences or transcripts of selected types of HPV. Target transcripts contain at least a portion of the ORF to allow for stable hybridization of the detector probes and at least one capture probe. Probes are designed in such a manner that efficiency of hybridization of probes to target transcripts is not diminished by excision of introns from transcripts (e.g. , formation of E6*I and E6*II) . 5. The integrity of probes used is maintained in the conditions of the assay. Oligonucleotide probes which are particularly useful as capture probes in the present invention are poly- (dA) -tailed oligonucleotide probes, such as those prepared according to the method described by M.L. Collins in European Patent Application 0265244, filed October 21, 1987, the teachings of which are incorporated herein by reference. Capture probes which are prehybridized to a solid substrate also can be used. Prehybridized probes are described in detail by M.L. Collins et al. in co-pending U.S. patent application number 07/321,728 filed March 10, 1989, the teachings of which are incorporated herein by reference. The second type of probe used in the present method is a riboprobe or detector probe whose sequence is specific for an HPV transcript or selected HPV DNA, and which generally have a de¬ tectable label, such as a radionuclide , or fluores¬ cent indicator. Detector probes are used to quanti- tate the DNA or mRNA. Detector probes can be prepared by art recognized techniques. Detector probes are generally single stranded RNA probes which hybridize to bi- or polycistronic transcripts of a selected HPV gene, such as the E6 or E7 gene. The detector probes are preferably labeled with a 32 125 radionuclide , such as P or I, but can be labeled by any means which does not interfere with the ability of the detector probes to hybridize to complementary sequences and which can be detected using available techniques. The present assay system and method of using it can be incorporated into a kit for clinical use. Such a kit includes a sample processing solution containing a chelating agent, such as EDTA, and an agent for disrupting molecular structures (e.g. , membranes) of cells (e.g. , a chaotropic agent); the tailed capture probes and labelled detector probes; at least one buffer; an agent for inhibiting RNAase enzymes (to prevent degradation of the target transcripts); a solid support (e.g. , magnetic heads or polystyrene substrate) coated with a polynucleo- tide which is complementary to the capture probe tail; reference or standard nucleic acids to be run simultaneously with the sample as a control; and a wash buffer containing a detergent and an agent (e.g. , a chaotropic agent) for disrupting molecular structures of cells. The kit can optionally contain additional wash buffers, a means for detecting the labelled detector probe, one or more elution buffers, amplification or cloning reagents and/or additional positive control samples or negative control sample. Amplification of the target sequences, if desired, can be accomplished, for example, by the technique described by Mullis in U.S. Patent 4,683,202. Amplification of the detector probe, after it has been cycled with the hybridization complexes, and/or cloning of the target sequences, can be accomplished, for example, by the method described by Maniatis et_aTL . in M£1e uiar_C_i£ning A_Lab£rat£ry_ManuaT ■ Cold Sprin; Harbor Laboratory, Cold Spring Harbor, NY (1982) . The invention will now be further illustrated by the following Exemplification. EXEMPLIFICATiON De jgn_ j Pre£arat £n j __a.nd_Phχ £ l_De £ri£ti£n_£f Pr££ HPV type-specific oligonucleotide capture probes, about 40 nucleotides long are shown in Figure 3. The Wordsearch/Segments Programs of the University of Wisconsin Suite was used to identify regions of HPV genomic , sense strands (i.e. , RNA sense) which might hybridize to these 33 oligonucleotide probes, and results of these analyses are shown in the Table. Table. Predicted Number of Base Pairs Between Oligonucleotide Probes and Discreet Regions of HPV Genomes HPV Probe Probe ORF Nts Recognized 6 11 16 18 31 33 35 Numbers in the Table refer to the base pairs in a discreet region of the viral genome which are predicted, from computer analyses (algorithm of Wilbur and Lipman; P_-.N^ ^S^ £0:726-739, 1983)) , to pair with portions of the oligonucleotides . Numbers after slash marks (/) indicate the gaps that would be needed in the oligonucleotide or target sequence to allow for the number of base pairs. For each HPV type, the nucleotide sequences (GenBank designa¬ tions) that are covered by each type-specific oligonucleotide are shown. In some cases, the computer program did not find any likely regions of significant base pairing between the oligonucleotide sequences and genomic sequences; these cases are noted "not found" (NF) . The oligonucleotides in Table 1 are unlikely to form stable hybrids with other types of HPV, therefore, they are considered to be type-specific . Design of Exon-Specific Capture_Probes_for_HPV_16^ ΣY_i_i J ._HP _31 J __HPV_33_and_HPV_35 Splice junction sequences in the E6 ORF have been identified in several oncogenic types of HPV, including HPV 16, HPV 18, HPV 31, HPV 33, and HPV 35 but they are not present in the E6 ORF's of HPV 6 and HPV 11. These splice donor and acceptor sites are utilized in HPV 16 and HPV 18, as is demonstrated by the presence of spliced RNA's (determined by sequencing cDNA's) in carcinoma cell lines. The presence of spliced transcripts can be used as predictors of progression to more serious disease. Thus, splice-junction capture probes which are predicted to form stable hybrids only with spliced transcripts were designed. Approximately 20 nucleotides of each capture probe hybridize to the 3' end of the upstream exon and the remaining 20 nucleotides hybridize to the 5' end of the downstream exon. Sequences of these probes and their target sites on transcripts are shown in Figure 4. Analyses of the alignment (algorithm of Wilbur and Lipman) of splice-junction probes and the genomes of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, and HPV 33 revealed that each probe is predicted to hybridize only to the transcripts of the HPV type for which the probe was designed. Different probes were designed for detection of each of the two E6 spliced mRNA's (E6*I and E6*II) of HPV 16. De ign_£f_HPy_ ^S>£e£if £_Ca£ U£ _Pr£be _and ^Σ∑ang me _£f_Pr£be _in_th£_HPV_i6_A aγ Ten HPV 16-specific oligonucleotide probes (about 40 nucleotides long) were designed and tested in the assay, described below. Three of these probes, designated 16-3, 16-4, and 16-5, were adapted for use in the assay because they afforded high sensitivity and specificity. The placement of these three probes with respect to the RNA target and the detector probes is shown in Figure 5. Capture probe 16-3, the probe closest to the detector probe, hybridizes to target sequences 46 nucleotides beyond the 3' end of the hybridization site covered by the detector riboprobe. Therefore, after hybridization of the detector probe to mRNA, only a short portion remains single stranded (i.e. , not covered by capture or detector probes), thus minimizing the chances for degradation by RNases. The alignment of these capture probes with sequences in HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33 and HPV 35 is shown in Figure 6. Inspection of potential base pairs between these probes and the other genital HPV types, an alignment that was made by visual and computer analyses, reveals that base mismatches (not underlined) are numerous and ap¬ proximately evenly distributed along the capture probes. As a result, these hybrids would be very unstable . The 3' ends of capture probes are extended by sequential addition of d(A) residues with terminal transferase such that the final poly-d(A) "tail" is 150-200 nucleotide long, the minimum length that will allow unencumbered capture of the hybridization complex to d(T) -modified magnetic beads. 2££i££_££^__P£ £ £i££_£ _M _i^ . ££i £_D _£ ££££ Ei_^£EΣ£££_ £<ϊ_Target_RNA HPV 16 DNA was cloned from CaSki cells, known to contain HPV 16 genomes (Baker et al . , J_. Y r£i. , UliAl * 962-971, 1987), into lambda 47.1 from which it was subcloned into the pT7/T3α-18 plas id (Bethesda Research Laboratory) . A summary of the cloning/subcloning scheme is shown in Figure 7. A 630 nucleotide Dde I fragment (nucleotides 25-655; Seedorf et al . , Virol. , 145:181-185, 1985) was excised, end-filled, and subcloned into Sma I- restricted pT7/T3α-18 plasmid. Sequence analysis, obtained by double or triple reads in most cases, revealed that the insert contained only nts 25-655 of the HPV 16 genome. Iτ\ vitro transcription with T7 polymerase on this construct, linearized with Ssp I restriction endonuclease , results in a single product with a homogeneous electrophoretic mobility and an apparent size of 0.5 Kb, that should hybridize to the 3' three- fourths of the E6 ORF and the 5' third of the E7 ORF. The detector probe contains, in addition to 471 nucleotides of the HPV 16 sequence, 22 additional nucleotides of the plasmid sequence on its 5' end, a result of transcription initiation on the T7 promoter and procession through a portion of the multiple cloning site in the vector. These additional 5' nucleotides will not interfere with hybridization of the capture probes, the most proximal being 46 nucleotides away. The detector probe, after hybridization to target mRNA, should extend about 40 nucleotides 5' to the splice donor site at nucleotides 224-232 (Figure 5) ; this hybrid is predicted to be very stable. Excision of either of the E6 introns would result in mRNA molecules that base pair with either 162 or 288 nucleotides of the detector probes . These hybrids should be stable in the assay. Following the manufacturers' instructions for synthesis of 32 transcripts with T7 polymerase, P-labeled o riboprobes with specific activities of 5 x 10 cpm/μg are produced. Placement of the detector probe on E6/E7 transcripts was designed such that E6/E7 target sequences flanking the hybridization site of the detector riboprobe would be available for hybridization to capture probes. The sensitivity of the HPV 16 assay was also determined. For this purpose it was important to produce, by i£_vitr£ transcription, well characterized positive targets (i.e. , mRNA-like molecules which contain the ORF's of the E6 and E7 genes). Transcription from the T3 promoter in the construct which contained full length genomic HPV 16, revealed a putative transcription terminator just upstream from the E6 and E7 genes (nucleotides 7400-7600 in the non-coding region) . Visual inspection of the sequences in this region, which lies just downstream from a polyadenylation signal, suggests that transcription of plus-sense RNA from this portion of the genome would produce RNA's which have numerous stretches of U residues, character¬ istics of transcription terminator; therefore, a 1.3 Kb Sph I fragment which includes about half of the region rich in termination sequences and a few sequences in the vector was deleted. Transcription from the resulting construct (linearized with Spe I) with T3 polymerase yields one expected major transcript of 1.9 Kb as well as a shorter minor RNA species (about 600 nucleotides shorter than the major one). The 1.9 Kb transcript was used as a positive target in the assay. - £ ££_ -^_ . E -E_l£_£3££££_£_ __£^_ : _SZ _i6._ s_ay Preparation of samples and procedures for performance of the assay are the same as those described by M. Collins in European Patent Application, Number 0265244, the teachings of which are incorporated herein by reference. In brief, cells from cervical specimens (e.g. , cervical lavage , scrapes, bioposies, and tumors) are mixed with sample processing buffer (SPB), containing 5M guanidinium isothiocynate (GuSCN) and 0.1M ethylenediaminetetraacetic acid (EDTA) in order to solubilize the cells and sample matrix and to inhibit RNAses. Samples are diluted such that the final concentration of GuSCN is 2.5M and incubated 7 with 67 ng/ml of each capture probe and 1.4 x 10 cpm/ml of detector probe at 37° for 2-18 hours. Then, d(T) -coated magnetic beads are added (final concentration of 0.125% solids) and the resulting mixture is incubated at 37" for 15 minutes; d(A) -tailed capture probes hybridize to the d(T) -coated magnetic beads during this period. Wash buffer (0.5 M GuSCN, 40 mMTris-HCl, pH 7.8, 10 m MEDTA, 0.5% sarkosyl, 0.2% bovine serum albumin, and 0.1% antifoam) is added to the mixture and the magnetic beads then are concentrated on the walls of the tubes by a magnetic field. The supernatant fluid is aspirated. Beads are washed two additional times with wash buffer, following the procedure just described, and then re-suspended in a buffer containing 3.25M GuSCN, lOOmM Tris-HCl, pH 7.8, 65mM EDTA, 0.5% BSA, and 0.5% sarkosyl. Following incubation of the suspension at 37" for 10 minutes, nucleic acid complexes should be released from the beads due to disruption of the A-T bonds between the poly-d(A) tails of .he capture probes and the oligo-d(T) chains of the magnetic beads. After concentration of the magnetic beads on the tube walls by a magnetic field, the release buffer and nucleic acid complexes are transferred to another tube and hybridization complexes are captured on a second set of magnetic beads, following the procedure just described. This capture, release, and re-capture of the nucleic acid complexes on magnetic beads is called reversible target capture. A second round of reversible target capture is performed and nucleic acid complexes in the release buffer are captured on a third set of beads . After the beads are washed once , they are resuspended in wash buffer and an aliquot of the suspension is applied to a nitrocellulose membrane. Radioactivity can be detected by exposure of the membrane to film or by quantitation of counts on a beta emission blot analyzer (e.g. , Betagen) . Exposure of the film may be quantitated on a densitometer. Alternatively, fluorescein-labeled detector oligonucleotides or riboprobes may be used in the assay. Efforts were made to quantitate the sensitivity of the HPV 16 assay using i * n._vitr£-generated posi¬ tive target transcripts. Examination of the X-ray film revealed (Figure 8A) that 500 fg of target (500,000 molecules) could be visualized and that a signal was not detected if target RNA was omitted; no signal was detected if lysates from 200,000 HeLa cells (which contain HPV 18 sequences) are used in the assay. A standard curve, produced by plotting radioactivity on beads vs. amount of positive target RNA, is a straight line for RNA target values of 100 fg to 100 pg (Figure 8B).

Claims: CLAIMS 1. A method of detecting in a sample obtained from an individual at least one high-oncogenic type of HPV, comprising the steps of: a) rendering nucleic acids present in the sample available for hybridization with complementary nucleotide sequences; b) combining the product of step (a) with at least one nucleic acid probe having a nucleotide sequence complementary to a gene of a high-oncogenic type HPV, wherein said gene is selected from the group consisting of the E6 gene, the E7 gene and spliced transcripts of the E6 or E7 gene, or both, under conditions appropriate for hybridization of complementary nucleotide sequences to occur; and c) detecting hybridization of the complemen¬ tary nucleotide sequences . 2. The method of Claim 1 wherein the high-onco¬ genic type HPV is selected from the group consisting of HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52 and HPV 56. 3. The method of Claim 2 further comprising quantitating at least one high-oncogenic type HPV, wherein in step (b) , the extent to which hybridization of complementary nucleotide sequences occurs is determined. 4. The method of Claim 1 further comprising combining the product of step (a) with a set of nucleic acid probes, the set comprising at least two different nucleic acid probes, each of said probes having a nucleotide sequence complementary to a different selected gene or transcript of a high-oncogenic type HPV. 5. The method of Claim 4 further comprising quantitating in step (c) the extent to which hybridization of each of the nucleotide se¬ quences occurs . 6. A method of detecting, in a cervical sample, a transcript of at least one high-oncogenic type HPV, comprising the steps of: a) combining the cervical sample, previously treated to render nucleic acids present in the sample available for hybridization with complementary nucleotide sequences, with at least one nucleic acid probe having a nucleotide sequence complementary to the E6 gene, E7 gene or spliced transcripts of the E6 or E7 gene, or both, of a high-oncogenic type HPV under conditions appropriate for hybridization of complementary nucleotide sequences to occur; and b) detecting hybridization of the complemen¬ tary nucleotide sequences. A method for detecting, in a cervical sample, a target nucleotide sequence which is the trans¬ cript of a gene of at least one selected high-oncogenic type HPV, comprising the steps of: a) treating the cervical sample to render nucleic acids present in the sample available for hybridization with complementary nucleotide sequences ; b) contacting the product of step (a) with: 1) at least one tailed capture probe which a) is complementary to a transcript of at least a portion of the E6 or E7 gene, the gene of the selected high-oncogenic type HPV; and b) has a polynucleotide tail; and 2) a labeled detector probe which is complementary to a different portion of the transcript of the E6 or E7 gene of the selected high-oncogenic type HPV, under conditions appropri¬ ate for hybridization of comple¬ mentary nucleotide sequences to occur, thereby forming a capture probe/ target nucleotide sequence/ detector probe hybridization complex; c) contacting the product of step (b) with a solid support having affixed thereto a polynucleotide which is complementary to the polynucleotide tail of the capture probe present in the hybridization com¬ plex, under conditions appropriate for hybridization of complementary nucleotide sequences to occur, thereby forming a hybridization complex/solid support complex; d) separating the hybridization complex/solid support complex from the product of step (c) by removing the solid support; and e) detecting the hybridization complex/solid support complex, the presence of which is indicative of the presence in the cervical sample of the target nucleotide sequence. 8. The method of Claim 7 wherein the target nucleotide sequence comprises a nucleotide sequence selected from the group consisting of: a transcript of the E6 gene of a high-oncogenic type HPV gene; a spliced transcript of the E6 gene of a high-oncogenic type HPV gene; a transcript of the E7 gene of a high-oncogenic type HPV gene; and a combination thereof. 9. The method of Claim 8 wherein the high- oncogenic type HPV is selected from the group consisting of: HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52 and HPV 56. 10. The method of Claim 9 wherein the target nucleotide sequence further comprises HPV nucleotide sequences downstream of the E7 gene. 11. The method of Claim 9 wherein the capture probe is selected from the group consisting of: probes 16-1, 16-2, 16-3, 16-4, 16-5, 18-1, 18-2, 18-3, 18-4, 18-5, 18-6, 31-1, 31-2, 31-3, 31-4, 31-5, 33-1, 33-2, 33-3, 33-4, 33-5 and 33-6, 35-1, 35-2, 35-3, 35-4 and 35-5, as represented in Figure 3. 12. The method of Claim 9 wherein the capture probe is complementary to a spliced transcript of the E6 gene of a high-oncogenic type HPV, said probe selected from the group consisting of: ExHPV16E6*I, ExHPVl6E6*II , ExHPV18E6*, ExHPV31E6*. ExHPV33E6*. and ExHPV35E6* as represented in Figure 4. 13. The method of Claim 9 wherein the polynucleotide tail of the capture probe is poly d(A) or poly d(T) . 14. The method of Claim 9 wherein the detector probe is labeled with a radionuclide. 15. The method of Claim 14 wherein the radionuclide , 32 D 125- is P or I. 16. The method of Claim 9 wherein the solid support is selected from the group consisting of: paramagnetic beads, polystyrene beads and poly¬ styrene dipsticks. 17. The method of Claim 16 wherein the solid support has affixed thereto a polynucleotide selected from the group consisting of poly d(T) and poly d(A) . 18. The method of Claim 17 wherein the capture probe is prehybridized with the polynucleotide affixed to the solid substrate. 19. The method of Claim 7 further comprising in step (b) contacting the product of step (a) with a set of nucleic acid probes, the set comprising at least two different nucleic acid probes, each of said probes being a nucleotide sequence complementary to all or a portion of a selected gene of a high-oncogenic type HPV. 20. The method of Claim 7 further comprising quantitating the target nucleotide sequence, wherein in step (f) , the extent to which hybridization of complementary nucleotide sequences occurs is determined. 21. A kit for detecting or quantitating, in a cervical sample, a target nucleotide sequence of at least one high-oncogenic type HPV, the target nucleotide sequence being either DNA or RNA, comprising: a) a chelating agent; b) an agent for disrupting molecular struc¬ tures in cells; c) at least one capture probe which a) is complementary to the target nucleotide sequence and b) has apolynucleotide tail; d) at least one detector probe which is a nucleotide sequence complementary to at least a portion of the target nucleotide sequence ; e) at least one buffer; f) an agent for inhibiting RNAase enzymes; g) a solid support having affixed thereto a polynucleotide complementary to the polynucleotide tail of the capture probe; h) reference nucleic acids ; and I) a detergent. 22. The kit of Claim 21 wherein the chelating agent is EDTA or DTPA. 23. The kit of Claim 22 wherein the agent for disrupting molecular structures In cells is a chaotropic agent. 24. The kit of Claim 23 wherein the polynucleotide tail of the oligonucleotide capture probes is a d(A) tail or d(T) tail. 25. The kit of Claim 24 wherein the detector probe is labelled. 26. The kit of Claim 25 wherein the solid support is selected from the group consisting of magnetic beads, polystyrene beads and poly¬ styrene dipsticks. 27. A method of determining the risk of progression to serious dysplasia of the cervix or to cervical carcinoma in a female in whom HPV is present in the cervix, comprising detecting, in a cervical sample, at least one target nucleotide sequence which Is all or a portion of a gene of an HPV type or a transcript thereof selected from the group consisting of the E6 gene of a high oncogenic type of HPV, the E7 gene of a high oncogenic type HPV, a spliced transcript of the E6 gene or the E7 gene, or both, of a high oncogenic type of HPV and a combination thereof, and comparing the results with the patterns of transcripts which are associated with high grade dysplasia or carcinoma in situ, or with the risk of progression to serious dysplasia of the cervix or to cervical carcinoma. 28. The method of Claim 27 wherein the high- oncogenic type HPV is selected from the group consisting of: HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52 and HPV 56. 29. The method of Claim 27 further comprising determining the quantity of the target nucleo¬ tide sequence in the cervical sample and comparing the quantity of target nucleotide sequence in the sample with the patterns of transcripts which are associated with high grade dysplasia or carcinoma in situ or with risk of progression to serious dysplasia of the cervix or to cervical carcinoma. 30. The method of Claim 29 further comprising detecting in a cervical sample from the female at least two different target nucleotide sequences, all of said target nucleotide sequences being either DNA of a high-oncogenic type HPV or a transcript thereof, but not a mixture thereof. 31. A nucleic acid probe comprising a nucleotide sequence selected from the group consisting of nucleotide sequences complementary to the nucleotide sequence of at least a region of the E6 gene of an high-oncogenic type HPV and nucleotide sequences complementary to the nucleotide sequence of at least a region of the E7 gene of high-oncogenic type HPV. 32. A nucleic acid probe of Claim 31 wherein the nucleotide sequence comprises at least a portion of a nucleotide sequence selected from the group consisting of the nucleotide se¬ quences of probes 16-1, 16-2, 16-3, 16-4, 16-5, 18-1, 18-2, 18-3, 18-4, 18-5, 18-6, 31-1, 31-2, 31-3, 31-4, 31-5, 33-1, 33-2, 33-3, 33-4, 33-5 33-6, 35-1, 35-2, 35-3, 35-4 and 35-5, as represented in Figure 3. 33. A nucleic acid probe of Claim 32 further comprising a polynucleotide tail. 34. A nucleic acid probe of Claim 33 wherein the polynucleotide tail is poly d(A) or poly d(T) . 35. A nucleic acid probe consisting essentially of a nucleotide sequence complementary to all or a portion of the E6 gene of a HPV type selected from the group consisting of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52 and HPV 56. 36. A nucleic acid probe consisting essentially of a nucleotide sequence complementary to all or a portion of the E7 gene of a HPV type selected from the group consisting of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52 and HPV 56. 37. A nucleic acid probe comprising a nucleotide sequence complementary to the nucleotide sequence of a spliced transcript of at least a region of the E6 gene and at least a region of the E7 gene of a high-oncogenic type HPV. 38. A nucleic acid probe of Claim 37 selected from the group consisting of: ExHPV16E6*I, ExHPV16E6*II, ExHPV18E6*. ExHPV31E6*. ExHPV33E6*, and ExHPV35E6*, as represented in Figure 4.

Inventors: HENDRICKS DAVID A, LANE DAVID J, PARODOS KYRIAKI, RIGBY SUSAN, HENDRICKS, DAVID, A., LANE, DAVID, J., PARODOS, KYRIAKI, RIGBY, SUSAN
Applicants: GENE TRAK SYSTEMS, GENE-TRAK SYSTEMS
Application year: 1990
Main classification: C12Q1
IPC codes: C12N15, C12Q1