US009371569B2

(12) United States Patent (10) Patent No.: US 9,371,569 B2 Van Criekinge et al. (45) Date of Patent: Jun. 21, 2016

(54) DETECTION AND PROGNOSIS OF (52) U.S. Cl. CERVICAL CANCER CPC ...... CI2O I/6886 (2013.01); C12O 2600/1 12 (2013.01); C12O 2600/154 (2013.01); C12O (71) Applicant: MDxHealth SA, Sart-Tilman (BE) 2600/16 (2013.01) (58) Field of Classification Search (72) Inventors: Wim Van Criekinge, Sart-Tilman (BE); None Valerie Deregowski, Sart-Tilman (BE). See application file for complete search history. Luc Dehaspe, Sart-Tilman (BE); G. Bea A. Wisman, Groningen (NL); Ate G. J. (56) References Cited Van der Zee, Groningen (NL); E. M. D. Schuuring, Groningen (NL) U.S. PATENT DOCUMENTS 2005/0250137 A1* 1 1/2005 Tainsky et al...... 435/6 (73) Assignee: MDxHealth S.A., Herstal (BE) 2010/0.14483.6 A1* 6/2010 Van Engeland et al. ... 514/44 A (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. WO WO 2006113678 A2 * 10, 2006 OTHER PUBLICATIONS (21) Appl. No.: 14/180,239 Widschwendter et al., Gynecologic Oncology, 2004 vol. 93 pp. 407 (22) Filed: Feb. 13, 2014 416. (65) Prior Publication Data * cited by examiner US 2015/OO17634 A1 Jan. 15, 2015 Primary Examiner — Michele KJoike Assistant Examiner — Mindy G Brown Related U.S. ApplicationO O Data Property(74) Attorney, Law, LLPAgent, or Firm — Andrus Intellectual (63) applicationContinuation No. of applicationPCT/EP2009/053386 No. 12/933,747, on Mar. filed 23, as (7) ABSTRACT 2009, now abandoned. The present invention relates to methods and kits for identi fying, diagnosing, prognosing, and monitoring cervical can (60) Eyal application No. 61/038,549, filed on Mar. cer. These methods include determining the methylation sta s tus or the expression levels of particular , or a (51) Int. Cl combination thereof. (2006.01) 23 Claims, 15 Drawing Sheets U.S. Patent Jun. 21, 2016 Sheet 1 of 15 US 9,371,569 B2

Figure 1A

U.S. Patent Jun. 21, 2016 Sheet 2 of 15 US 9,371,569 B2

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U.S. Patent Jun. 21, 2016 Sheet 4 of 15 US 9,371,569 B2

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U.S. Patent Jun. 21, 2016 Sheet 9 Of 15 US 9,371,569 B2

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U.S. Patent Jun. 21, 2016 Sheet 11 of 15 US 9,371,569 B2

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i US 9,371,569 B2 1. 2 DETECTION AND PROGNOSS OF absolute and its longevity is uncertain; as yet, the possibility CERVICAL CANCER of genotype replacement cannot be excluded; and older women not covered by vaccination programs will continue to FIELD OF THE INVENTION be at risk. Therefore, cervical screening will still be required for control. The present invention relates to the area of cancer diagnos Cancer biomarkers have been described in literature and tics and therapeutics. In particular, it relates to methods and aberrant methylation of genes has been linked to cervical kits for identifying, diagnosing, prognosing, and monitoring cancer (Virmani et al., 2001). In addition, methylation mark cervical cancer. These methods include determining the ers may serve for predictive purposes as they often reflect the methylation status or the expression levels of particular 10 sensitivity to therapy or duration of patient Survival. genes, or a combination thereof. DNA methylation is a chemical modification of DNA per formed by called methyltransferases, in which a BACKGROUND TO THE INVENTION methyl group (m) is added to certain cytosines (C) of DNA. Cervical cancer is the fifth most deadly cancer in women. 15 This non-mutational (epigenetic) process (mC) is a critical Worldwide, approximately 500,000 cases of cervical cancer factor in regulation. (See J. G. Herman, are diagnosed and about 250,000 women die from this disease Seminars in Cancer Biology, 9: 359-67, 1999). annually (www.who.int/mediacentre/factsheets). An early diagnosis is critical for the Successful treatment of Most (80-90%) invasive cervical cancer develops in flat, many types of cancer, including cervical cancer. If the exact scaly Surface cells that line the cervix (called squamous cell methylation profiles of cervical tumors are available and carcinomas, SCC). Approximately 10-15% of cases develop drugs targeting the specific genes are obtainable, then the in glandular Surface cells (called adenocarcinomas, AdC). treatment of cervical cancer could be more focused and ratio Less commonly, cervical cancers have features of both SCC nal. Therefore, the detection and mapping of novel methyla and AdC. These are called adenosquamous carcinomas or tion markers is an essential step towards improvement of mixed carcinomas (www.cancer.org). 25 cervical cancer prevention, Screening, and treatment. Thus, During the process of cervical cancer development, normal there is a continuing need in the art to identify methylation cervical cells gradually develop pre-cancerous changes that markers that can be used for improved assessment of cervical turn into cancer. Cervical cancer evolves from pre-existing CaCC. noninvasive premalignant lesions referred to as cervical intraepithelial neoplasias (CINs), ranging from CINI (mild 30 SUMMARY OF THE INVENTION dysplasia) to CIN II (moderate dysplasia) to CIN III (severe dysplasia/carcinoma in situ). This process usually takes sev The present invention is based on the finding that several eral years but sometimes can happen in less than a year. For genes are identified as being differentially methylated in cer most women, pre-cancerous cells will remain unchanged and Vical cancers. This information is useful for cervical cancer disappear without any treatment. 35 screening, risk-assessment, prognosis, disease identification, Screening for malignant and premalignant disorders of the disease staging, and identification of therapeutic targets. The cervix is usually performed according to the Papanicolaou identification of new genes that are methylated in cervical (PAP) system. The cervical smears are examined by light cancer allows accurate and effective early diagnostic assays, microscopy and the specimens containing morphologically methylation profiling using multiple genes and identification abnormal cells are classified into PAP I to V, at a scale of 40 of new targets for therapeutic intervention. increasing severity of the lesion. But, present PAP test has Accordingly, in a first aspect, the invention provides a Some limitations and is not completely ideal for screening as method for identifying cervical cancer or its precursor, or it suffers from suboptimal single-test sensitivity, limited predisposition to cervical cancer. Epigenetic modification of reproducibility, and many equivocal. at least one gene selected from the group consisting of genes There is a strong association between certain Subtypes of 45 according to Table 1, is detected in a test sample containing the Human Papillomavirus (HPV) and cervical cancer. Stud cervical cells or nucleic acids from cervical cells. The test ies have shown that only high-risk HPV types are involved in sample is identified as containing cells that are neoplastic, the progression from cytological normal cervix cells to high precursor to neoplastic, or predisposed to neoplasia, or as grade squamous intraepithelial lesions. Around 15 high-risk containing nucleic acids from cells that are neoplastic, pre (cancer-causing) HPV types have been identified. Although it 50 cursor to neoplastic, or predisposed to neoplasia. has been Suggested that high-risk HPV testing may improve Preferably, the at least one gene is selected from a group of cervical cancer Screening, the specificity for high grade cer genes consisting of JAM3. LMX1A, CDO1, NID2, ALX3, vical neoplasia of high risk HPV testing is relatively low. This ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, low specificity of HPV testing leads to a higher number of BMP6, BNIP3, C13orf18, C16orf248, C90rf19, CALCA, unnecessarily follow-up diagnostic workups (e.g. colpos 55 CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, copy) and unnecessarily treatment with cryotherapy or loop CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, electroSurgical excision procedure, which permanently alters CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, the cervix and have unknown consequences on fertility and EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, pregnancy. GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, To improve early detection, the combination of HPV and 60 HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, PAP tests is now approved by the FDA for screening women HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, 30 years of age and older. However, co-testing Substantially LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, increases the cost of Screening. NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, In the meanwhile, vaccines for preventing cervical cancer PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, have been developed and one has already been approved by 65 RARA, RASSF1A, RBP4. RECK, RPRM, SALL4, the FDA. But, immunization will only protect against HPV SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, types that are targeted by the vaccine; protection will not be SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, US 9,371,569 B2 3 4 TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, fied; if the gene is unmethylated or methylated to a lesser WIF1, WIT1, WT1, XRCC3, and ZGPAT. degree, it is decided that there is no need for cervical resec In one embodiment of the present invention, the detection tion. of epigenetic modification comprises detection of methyla tion of a CpG dinucleotide motif in the gene and/or promoter 5 SUMMARY OF THE FIGURES region of the gene; and/or detection of expression of mRNA of the gene. FIGS. 1A, B, and C: The number of probes (w) that is The invention also relates to a kit for assessing cervical retrieved using parameters X (number of P-calls in primary cancer or its precursor, or predisposition to cervical cancer in cancers for probe), y (number of P-calls in untreated cell-lines 10 for probe) and Z (number of P-calls in treated cell-lines for a test sample containing cervical cells or nucleic acids from probe). cervical cells. The kit comprises in a package: a reagent that FIG. 2: Step-plot to determine optimal number of probes (a) modifies methylated cytosine residues but not non-methy for further analysis. Step-plot of the number of retrieved lated cytosine residues, or that (b) modifies non-methylated known markers as a function of the position after relaxation cytosine residues but not methylated cytosine residues; and at 15 ranking (this is the number of selected probes after ranking). least one pair of oligonucleotide primers that specifically The step plot shows the actual (observed) number of markers. hybridizes under amplification conditions to a region of a If the markers were randomly distributed, one would expect gene selected from the group consisting of genes according to the profile, marked with expected (details in the text). The Table 1 and/or the aforementioned group of genes. The region trend of the observed markers versus the number of selected is preferably within about 10 kbp of said gene's transcription probes is indicated with dashed lines. start site. FIG. 3: (Hyper) methylation analysis of the promoter In a further aspect, the invention provides for oligonucle region (-430 to -5 of TSS) of the CCNA1 gene by COBRA otide primers and/or probes and their sequences for use in the and sequence analysis. methods and assays of the invention. A: Schematic representation of the restriction sites The invention also relates to screening protocols for the 25 (B: BstUI and T: TaqI) in the virtual hypermethylated BSP screening of woman for cervical cancer and the precursors nucleotide sequence after bisulfate treatment. Vertical bars thereof. Such method for cervical cancer Screening combines represent CG site, arrow represents TSS (retrieved from hr-HPV testing and methylation testing, or combines PAP Ensembl). tests with methylation testing. Methylation testing in Such B: Result of COBRA analysis of the BSP products of 10 screening method preferably detects the epigenetic modifica 30 tumor samples (T1-T10), in vitro methylated DNA as a posi tion of at least one gene selected from the group consisting of tive control (IV) and leukocyte DNA as a negative (unmethy JAM3. LMX1A, CDO1, NID2, ALX3, ALX4, AR, ARID4A, lated) control (L). ATM, AURKA, B4GALT1, BMP2, BMP6, BNIP3, C: Schematic representation of the sequencing results. C13orf18, C16orf248, C90rf19, CALCA, CAMK4, CCNA1, From each tumor, the BSP-products were cloned into TOPO CCND2, CDH1, CDH4, CDK6, CDKN1B, CDKN2B, 35 pCR4 (Invitrogen) and sequencing (BaseGlear) was per CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, CYCLIND2, formed on M13-PCR products of 7-9 independent clones. DAPK1, DBC1, DDX19B, DKK2, EGFR, EGR4, EPB41L3, Circles represent CG dinucleotides: the darker, the more FOS, FOXE1, GADD45A, GATA4, GDAP1L1, GNB4, clones at this site were methylated. GPNMB, GREM1, Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, FIG. 4: Representative COBRA on 3 gene promoters (SST, HOXA11, HOXA7, HOXD1, IGSF4, ISYNA1, JPH3, 40 AUTS2 and SYCP3). KNDC1, KRAS, LAMA1, LOC285016, LOX, LTB4R, A: Schematic representation of the restriction enzyme sites MAL, MTAP, MYO18B, NDRG2, NOL4, NPTX1, NPTX2, in the virtual hypermethylated BSP nucleotide sequence after OGFOD2, PAK3, PAX1, PDCD4, PHACTR3, POMC, bisulfate treatment. (B: BstUI, T: TaqI and H: Hinfl). Bars PRKCE, RAD23B, RALY, RARA, RASSF1A, RBP4, represent CG site and arrow is TSS (retrieved from Ensembl). RECK, RPRM, SALL4, SEMA3F, SLC5A8, SLIT1, SLIT2, 45 B: Result of COBRA analysis of BSP products of tumor SLIT3, SMPD1, SOCS1, SOX1, SOX17, SPARC, SPN, SST, samples (T1-T10) and 5 normal cervices (N1–N5), in vitro TAC1, TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, methylated DNA as a positive control (IV) and leukocyte UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT. Depen DNA as a negative (unmethylated) control (L); lane B is water dent on the outcome, the women screened for cervical cancer blank. is referred for colposcopy, or referred for hr-HPV and/or PAP 50 FIG.S: testing and/or methylation testing on a more regular basis. A: Position of the different primers relative to the TSS Epigenetic loss of gene function can be rescued by the use (transcription start site). Multiple primer designs are dis of DNA demethylating agents and/or DNA methyltransferase played by blue boxes and red boxes (final primer pairs inhibitors. Accordingly, the invention also provides for a retained for the assays). The exon of ALX4 is indicated in method for predicting the likelihood of successful treatment 55 green. The number of CpG count is spotted in blue over a or resistance to treatment of cancer with Such agent. If the region of 20Kb. gene is methylated, the likelihood of Successful treatment is B: List of sequences for the different primer sets, converted higher than if the gene is unmethylated, or methylated to a and unconverted amplicon sequences used in FIG. 5 A. lesser degree. Conversely, if the gene is unmethylated, or FIGS. 6A and B: Ranked methylation table from the Light methylated to a lesser degree, the likelihood of resistance to 60 cycler platform. 27 methylation profiles from cervical cancer treatment is higher than if the gene is methylated. samples (left) are compared against 20 normal tissue samples In a related aspect, epigenetic loss of gene function(s) can (right). Samples are shown along the X-axis where each ver identify the stage of the disease and from that the need of tical column represents the methylation profile of one indi treatment. Accordingly, the invention provides for a method vidual sample across the 63 different assays (Y-axis). Assays for predicting suitable treatment comprising determining the 65 demonstrating the best methylation discriminators between methylation status of a gene or a combination of genes. If the the 2 groups are displayed at the top, with discrimination gene is methylated, the need of cervical resection is identi effect decreasing towards the bottom. The black boxes indi US 9,371,569 B2 5 6 cate the methylated results; grey boxes indicate the unmethy RASSF1A, RBP4. RECK, RPRM, SALL4, SEMA3F, lated results; white boxes indicate invalid results. (NA: not SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, SOCS1, SOX1, applicable; NT: not tested) SOX17, SPARC, SPN, SST, TAC1, TERT, TFPI-2, TLL1, FIG. 7: Amplification plot for the standard curve for TNFAIP1, TRMT1, TWIST1, UGT1A1, WIF1, WIT1, WT1, TAC1 561.87 5 XRCC3, and ZGPAT. FIG. 8: Amplification plot for standard curve and samples Accordingly, in a first aspect, the invention provides a for TAC1 561.87 method for identifying cervical cancer or its precursor, or FIG. 9: Linear regression of standard curve for predisposition to cervical cancer. Epigenetic modification of TAC1 561.87 at least one gene selected from the group consisting of genes FIG.10: Decision tree for ratio determination 10 according to Table 1, is detected in a test sample containing FIG.11: Performance of the individual markers oncervical cervical cells or nucleic acids from cervical cells. The test tissue samples using qMSP. sample is identified as containing cells that are neoplastic, DETAILED DESCRIPTION OF THE INVENTION precursor to neoplastic, or predisposed to neoplasia, or as 15 containing nucleic acids from cells that are neoplastic, pre We describe a new sorting methodology to enrich for genes cursor to neoplastic, or predisposed to neoplasia. which are silenced by promoter methylation in human cervi Preferably, the at least one gene is selected from a group of cal cancer. The pharmacological unmasking expression genes consisting of JAM3. LMX1A, CDO1, NID2, ALX3, microarray approach is an elegant method to enrich for genes ALX4, AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, that are silenced and re-expressed during functional reversal BMP6, BNIP3, C13orf18, C16orf248, C90rf19, CALCA, of DNA methylation upon treatment with demethylating CAMK4, CCNA1, CCND2, CDH1, CDH4, CDK6, agents. However, such experiments are performed in in vitro CDKN1B, CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, (cancer) cell lines mostly with poor relevance when extrapo CTDSPL, CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, lating to primary cancers. To overcome this problem, we EGFR, EGR4, EPB41L3, FOS, FOXE1, GADD45A, incorporated data from primary cancer samples in the experi 25 GATA4, GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, mental design. A pharmacological unmasking microarray HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, approach was combined with microarray expression data of HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, primary cancer Samples. For the integration of data from both LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, cell lines and primary cancers, we developed a novel ranking NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, strategy, which combines reactivation in cell lines and no 30 PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, expression in primary cancer tissue. RARA, RASSF1A, RBP4. RECK, RPRM, SALL4, We also used a Genome-wide Promoter Alignment SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, approach with the capacity to define a further substantial SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, fraction of the cancer gene promoter CpG island DNA methy TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, lome. Markers clustering with known methylation markers 35 WIF1, WIT1, WT1, XRCC3, and ZGPAT. might indicate towards common mechanisms underlying the Preferably, at least one gene is selected from the group methylation event and thus identify novel genes that are more consisting of JAM3. LMX1A, CDO1, NID2, CCNA1, methylation-prone. HOXA11, GREM1 and TAC1. Preferably, epigenetic silenc Studies of the genes defined by the different approaches ing of a gene combination is detected and preferably selected will contribute to understanding the molecular pathways driv 40 from the group of gene combinations consisting of: ing tumorigenesis, provide useful new DNA methylation NID2 and HOXA11; biomarkers to monitor cancer risk assessment, early diagno JAM3, CDO1, HOXA11, and CCNA1; sis, and prognosis, and permit better monitoring of gene re JAM3 and HOXA11; expression during cancer prevention and/or therapy strate JAM3. HOXA11 and GREM1; g1eS. 45 JAM3, NID2, HOXA11 and CDO1; Using the aforementioned techniques, we have identified JAM3, TAC1, HOXA11, and CDO1; cytosines within CpG dinucleotides of DNA from particular JAM3. HOXA11, and CDO1; genes isolated from a test sample, which are differentially JAM3 and CDO1, methylated in human cervical cancertissue samples and nor JAM3 and NID2: mal cervical tissue control samples. The cancer tissues 50 NID2 and CDO1; samples are hypermethylated or hypomethylated with respect JAM3 and LMX1A to the normal samples (collectively termed epigenetic modi NID2 and LMX1A, and fication). The differential methylation has been found in JAM3, CDO1 and NID2 genomic DNA of at least one gene selected from the group “Identifying a disease or predisposition of disease is consisting of JAM3. LMX1A, CDO1, NID2, ALX3, ALX4, 55 defined herein to include detecting by way of routine exami AR, ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, nation, screening for a disease or pre-stadia of a disease, BNIP3, C13orf18, C16orfA8, C90rf19, CALCA, CAMK4, monitoring staging and the state and/or progression of the CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, disease, checking for recurrence of disease following treat CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, ment and monitoring the Success of a particular treatment. CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, 60 The identification can also have prognostic value, and the EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, prognostic value of the tests can be used as a marker of GDAP1L1, GNB4, GPNMB, GREM1, Gst-Pi, HHIP, HIN1, potential Susceptibility to cancer. HOOK2, HOXA1, HOXA11, HOXA7, HOXD1, IGSF4, The term “Epigenetic modification' can be described as a ISYNA1, JPH3, KNDC1, KRAS, LAMA1, LOC285016, stable alteration in gene expression potential that takes place LOX, LTB4R, MAL, MTAP, MYO18B, NDRG2, NOL4, 65 during development and cell proliferation, mediated by NPTX1, NPTX2, OGFOD2, PAK3, PAX1, PDCD4, mechanisms other than alterations in the primary nucleotide PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, sequence of a gene. Three related mechanisms that cause US 9,371,569 B2 7 8 alteration in gene expression are recognized: DNA methyla digestion, and bands that differ between two tissues identify tion, histone code changes and RNA interference. cDNAs of interest. Specific can be assessed using Epigenetic modification of a gene can be determined by any convenient method including immunoassays and any method known in the art. One method is to determine that immuno-cytochemistry but are not limited to that. Most such a gene which is expressed in normal cells or other control methods will employ antibodies, or engineered equivalents cells is less expressed or not expressed in tumor cells. Dimin thereof, which are specific for the particular or protein ished gene expression can be assessed in terms of DNA fragments. The sequences of the mRNA (cDNA) and proteins methylation status or in terms of expression levels as deter of the markers of the present invention are known in the art mined by their methylation status, generally manifested as and publicly available. hypermethylation. Conversely, a gene can be more highly 10 Alternatively, methylation-sensitive restriction endonu expressed in tumor cells than in control cells in the case of hypomethylation. This method does not, on its own, however, cleases can be used to detect methylated CpG dinucleotide indicate that the silencing or activation is epigenetic, as the motifs. Such endonucleases may either preferentially cleave mechanism of the silencing or activation could be genetic, for methylated recognition sites relative to non-methylated rec example, by Somatic mutation. One method to determine that 15 ognition sites or preferentially cleave non-methylated relative silencing is epigenetic is to treat with a reagent, such as DAC to methylated recognition sites. Non limiting examples of the (5'-deaZacytidine), or with a reagent which changes the his former are Aat II, Acc III, Ad I, AcII, Age I, AIu I, Asc I, Ase tone acetylation status of cellular DNA or any other treatment 1, AsiS I, Ban I, Bbe I, BsaAI, BsaEI I, BsiE I, BsiW I, BSrV affecting epigenetic mechanisms present in cells, and observe I, BssK 1, BstB I, BstNI, Bs I, CIa I, Eae I, Eag I, Fau I, Fse that the silencing is reversed, i.e., that the expression of the I, Hha I, mP1 I, HinC II, Hpa 11, Npy99 I, HpyCAIV. Kas I, gene is reactivated or restored. Mbo I, MIu I, Map.A 11. MspI, Nae I, Nar I, Not 1, Pml I, Pst Another means to determine epigenetic modification is to I, Pvu I, Rsr II, Sac II, Sap I, Sau3AI, Sfl I, Sfo I, Sgr AI, Sma determine the presence of methylated CpG dinucleotide ISnaB I, Tsc I, Xma I, and Zra I. Nonlimiting examples of the motifs in the silenced gene or the absence of methylation CpG latter are AccII, Ava I, BssHII, BstUI, HpaII, Not I, and Mho dinucleotide motifs in the activated gene. In one embodiment, 25 I. epigenetic modification of a CpG dinucleotide motif in the Alternatively, chemical reagents can be used that selec promoter region of the at least one gene selected from a group tively modify either the methylated or non-methylated form of genes according to Table 1 is determined. Methylation of a of CpG dinucleotide motifs. Modified products can be CpG island at a promoter usually prevents expression of the detected directly, or after a further reaction which creates gene. The islands can Surround the 5' region of the coding 30 products that are easily distinguishable. Means which detect region of the gene as well as the 3' region of the coding region. altered size and/or charge can be used to detect modified Thus, CpG islands can be found in multiple regions of a products, including but not limited to electrophoresis, chro nucleic acid sequence. The term “region' when used in ref matography, and mass spectrometry. Examples of Such erence to a gene includes sequences upstream of coding chemical reagents for selective modification include hydra sequences in a regulatory region including a promoter region, 35 zine and bisulfite ions. Hydrazine-modified DNA can be in the coding regions (e.g., exons), downstream of coding treated with piperidine to cleave it. Bisulfite ion-treated DNA regions in, for example, enhancer regions, and in introns. All can be treated with alkali. Other means for detection that are of these regions can be assessed to determine their methyla reliant on specific sequences can be used, including but not tion status. When the CpG distribution in the promoter region limited to hybridization, amplification, sequencing, and is rather scarce, levels of methylation are assessed in the 40 ligase chain reaction. Combinations of Such techniques can intron and/or exon regions. The region of assessment can be a be used as is desired. region that comprises both intron and exon sequences and The principle behind electrophoresis is the separation of thus overlaps both regions. Typically these reside near the nucleic acids via their size and charge. Many assays exist for transcription start site (TSS), for example, within about 10 detecting methylation and most rely on determining the pres kbp, within about 5 kbp, within about 3 kbp, within about 1 45 ence or absence of a specific nucleic acid product. Gel elec kbp, within about 750 bp, within about 500 bp, within 200 bp trophoresis is commonly used in a laboratory for this purpose. or within 100 bp. Once a gene has been identified as the target One may use MALDI mass spectrometry in combination of epigenetic modification in tumor cells, determination of with a methylation detection assay to observe the size of a reduced or enhanced expression can be used as an indicator of nucleic acid product. The principle behind mass spectrometry epigenetic modification. 50 is the ionizing of nucleic acids and separating them according Expression of a gene can be assessed using any means to their mass to charge ratio. Similar to electrophoresis, one known in the art. Typically expression is assessed and com can use mass spectrometry to detect a specific nucleic acid pared in test samples and control samples which may be that was created in an experiment to determine methylation normal, non-malignant cells. Either mRNA or protein can be (Tost, J. et al. 2003). measured. Methods employing hybridization to nucleic acid 55 One form of chromatography, high performance liquid probes can be employed for measuring specific mRNAS. chromatography, is used to separate components of a mixture Such methods include using nucleic acid probe arrays (e.g. based on a variety of chemical interactions between a Sub microarray technology, in situ hybridization, Northern blots). stance being analyzed and a chromatography column. DNA is Messenger RNA can also be assessed using amplification first treated with sodium bisulfite, which converts an unm techniques, such as RT-PCR. Sequencing-based methods are 60 ethylated cytosine to uracil, while methylated cytosine resi an alternative; these methods started with the use of expressed dues remain unaffected. One may amplify the region contain sequence tags (ESTs), and now include methods based on ing potential methylation sites via PCR and separate the short tags, such as serial analysis of gene expression (SAGE) products via denaturing high performance liquid chromatog and massively parallel signature sequencing (MPSS). Differ raphy (DHPLC). DHPLC has the resolution capabilities to ential display techniques provide another means of analyzing 65 distinguish between methylated (containing cytosine) and gene expression; this family oftechniques is based on random unmethylated (containing uracil) DNA sequences. Deng, D. amplification of cDNA fragments generated by restriction et al. describes simultaneous detection of CpG methylation US 9,371,569 B2 10 and single nucleotide polymorphism by denaturing high per nucleotides, to the target, then the primer may also contain formance liquid chromatography. additional nucleotide residues that do not interfere with Hybridization is a technique for detecting specific nucleic hybridization but may be useful for other manipulations. acid sequences that is based on the annealing of two comple Exemplary of such other residues may be sites for restriction mentary nucleic acid strands to form a double-stranded mol endonuclease cleavage, for ligand binding or for factor bind ecule. One example of the use of hybridization is a microarray ing or linkers or repeats. The oligonucleotide primers may or assay to determine the methylation status of DNA. After may not be such that they are specific for modified methylated sodium bisulfite treatment of DNA, which converts an unm residues. ethylated cytosine to uracil while methylated cytosine resi One way to distinguish between modified and unmodified dues remain unaffected, oligonucleotides complementary to 10 DNA is to hybridize oligonucleotide primers which specifi potential methylation sites can hybridize to the bisulfite cally bind to one form or the other of the DNA. After primer treated DNA. The oligonucleotides are designed to be com hybridization, an amplification reaction can be performed. plimentary to either sequence containing uracil (thymine) or The presence of an amplification product indicates that a sequence containing cytosine, representing unmethylated sample hybridized to the primer. The specificity of the primer and methylated DNA, respectively. Computer-based microar 15 indicates whether the DNA had been modified or not, which ray technology can determine which oligonucleotides hybrid in turn indicates whether the DNA had been methylated or ize with the DNA sequence and one can deduce the methyla not. For example, bisulfite ions convert non-methylated tion status of the DNA. Similarly primers can be designed to cytosine bases to uracil bases. Uracil bases hybridize to be complimentary to either sequence containing uracil (thym adenine bases under hybridization conditions. Thus an oligo ine) or sequence containing cytosine. Primers and probes that nucleotide primer which comprises adenine bases in place of recognize the converted methylated form of DNA are dubbed guanine bases would hybridize to the bisulfite-modified methylation-specific primers or probes (MSP). DNA, whereas an oligonucleotide primer containing the gua An additional method of determining the results after nine bases would hybridize to the non-converted (initial sodium bisulfite treatment involves sequencing the DNA to methylated) cytosine residues in the modified DNA. Ampli directly observe any bisulfite-modifications. Pyrosequencing 25 fication using a DNA polymerase and a second primer yield technology is a method of sequencing-by-synthesis in real amplification products which can be readily observed. This time. It is based on an indirect bioluminometric assay of the method is known as MSP (Methylation Specific PCR; U.S. pyrophosphate (PPi) that is released from each deoxynucle Pat. Nos. 5,786,146; 6,017,704; 6,200,756). Primers are otide (dNTP) upon DNA-chain elongation. This method pre designed to anneal specifically with the converted sequence sents a DNA template-primer complex with a dNTP in the 30 representing either the methylated or the unmethylated ver presence of an exonuclease-deficient Klenow DNA poly sion of the DNA. Preferred primers and primer sets for assess merase. The four nucleotides are sequentially added to the ing the methylation status of the concerned gene by way of reaction mix in a predetermined order. If the nucleotide is MSP will specifically hybridize to a converted sequence pro complementary to the template base and thus incorporated, vided in Table 2, or to its complement sequence. Most pre PPi is released. The PPi and other reagents are used as a 35 ferred primers and primer sets are provided in Table 1 and are Substrate in a luciferase reaction producing visible light that is represented by SEQID NO.1 to 264. Sense primers comprise detected by either aluminometer or a charge-coupled device. or consist essentially of SEQ ID NO. 1 to 132, antisense The light produced is proportional to the number of nucle primers consist essentially of SEQ ID NO. 133 to 264. The otides added to the DNA primer and results in a peak indicat amplification products can be optionally hybridized to spe ing the number and type of nucleotide present in the form of 40 cific oligonucleotide probes which may also be specific for a program. Pyrosequencing can exploit the sequence differ certain products. Alternatively, oligonucleotide probes can be ences that arise following sodium bisulfite-conversion of used which will hybridize to amplification products from DNA. both modified and non-modified DNA. A variety of amplification techniques may be used in a Thus, present invention provides for a method for identi reaction for creating distinguishable products. Some of these 45 fying cervical cancer or its precursor, or predisposition to techniques employ PCR. Other suitable amplification meth cervical cancer in a test sample containing cervical cells or ods include the ligase chain reaction (LCR) (Barringer et al. nucleic acids from cervical cells comprising: contacting a 1990), transcription amplification (Kwoh et al. 1989; WO88/ methylated CpG-containing nucleic acid of at least one gene 10315), selective amplification of target polynucleotide selected from the group consisting of genes according to sequences (U.S. Pat. No. 6,410,276), consensus sequence 50 Table 1 with bisulfite to convert unmethylated cytosine to primed polymerase chain reaction (U.S. Pat. No. 4437,975), uracil; detecting the methylated CpGs in the nucleic acid by arbitrarily primed polymerase chain reaction (WO90/06995), contacting the converted nucleic acid with oligonucleotide nucleic acid based sequence amplification (NASBA) (U.S. primers whose sequence discriminates between the bisulfite Pat. Nos. 5,409,818; 5.554,517; 6,063,603), microsatellite treated methylated and unmethylated version of the converted length polymorphism (MLP), and nick displacement ampli 55 nucleic acid; and identifying the test sample as containing fication (WO2004/067726). cells that are neoplastic, precursor to neoplastic, or predis Sequence variation that reflects the methylation status at posed to neoplasia, or as containing nucleic acids from cells CpG dinucleotides in the original genomic DNA offers two that are neoplastic, precursor to neoplastic, or predisposed to approaches to PCR primer design. In the first approach, the neoplasia. primers do not themselves cover or hybridize to any potential 60 Modified and non-modified DNA can be distinguished sites of DNA methylation; sequence variation at sites of dif with use of oligonucleotide probes which may also be specific ferential methylation are located between the two primers. for certain products. Such probes can be hybridized directly Such primers are used in bisulfite genomic sequencing, to modified DNA or to amplification products of modified COBRA, Ms-SNuPE. In the second approach, the primers are DNA. Probes for assessing the methylation status of the con designed to anneal specifically with either the methylated or 65 cerned gene will specifically hybridize to the converted unmethylated version of the converted sequence. If there is a sequence but not to the corresponding non converted Sufficient region of complementarity, e.g., 12, 15, 18, or 20 sequence. Probes are designed to anneal specifically with the US 9,371,569 B2 11 12 converted sequence representing either the methylated or erably selected from the group of JAM3. LMX1A, CDO1, unmethylated version of the DNA. Preferred converted NID2, CCNA1, HOXA11, GREM1 and TAC1. Preferred sequences are provided in Table 2. Preferred probes anneal gene combinations include specifically with the converted sequence representing the NID2 and HOXA11; methylated version of the DNA, or to the complement JAM3, CDO1, HOXA11, and CCNA1; sequence thereof. Oligonucleotide probes can be labeled JAM3 and HOXA11; using detection systems known in the art. These include but JAM3. HOXA11 and GREM1; are not limited to fluorescent moieties, radioisotope labeled JAM3, NID2, HOXA11 and CDO1; moieties, bioluminescent moieties, luminescent moieties, JAM3, TAC1, HOXA11, and CDO1; chemiluminescent moieties, enzymes, Substrates, receptors, 10 JAM3. HOXA11, and CDO1; or ligands. JAM3 and CDO1, JAM3 and NID2: Another way for the identification of methylated CpG NID2 and CDO1; dinucleotides utilizes the ability of the MBD domain of the JAM3 and LMX1A McCP2 protein to selectively bind to methylated DNA 15 NID2 and LMX1A, and sequences (Cross et al., 1994; Shiraishi et al., 1999). Restric JAM3, CDO1 and NID2. tion endonuclease digested genomic DNA is loaded onto The accession numbers corresponding to the listed genes expressed His-tagged methyl-CpG binding domain that is can be found at the website for the National Center for Bio immobilized to a solid matrix and used for preparative col technology Information. Ofcourse, as appropriate, the skilled umn chromatography to isolate highly methylated DNA person would appreciate that functionally relevant variants of sequences. Variants of this method have been described and each of the gene sequences may also be detected according to may be used in present methods of the invention. the methods of the invention. For example, the methylation Real time chemistry allows for the detection of PCR ampli status of a number of splice variants may be determined fication during the early phases of the reactions, and makes according to the methods of the invention. Variant sequences quantitation of DNA and RNA easier and more precise. A few 25 preferably have at least 90%, at least 91%, at least 92%, at variants of real-time PCR are well known. They include Taq least 93%, at least 94%, at least 95%, at least 96%, at least man(R) (Roche Molecular Systems), Molecular Beacons(R), 97%, at least 98%, or at least 99% nucleotide sequence iden Amplifluor R (Chemicon International) and Scorpion(R) tity with the nucleotide sequences in the database entries. DzyNAR), PlexorTM (Promega) etc. The TaqMan(R) system Computer programs for determining percentage nucleotide and Molecular Beacon R) system have separate probes labeled 30 sequence identity are available in the art, including the Basic with a fluorophore and a fuorescence quencher. In the Scor Local Alignment Search Tool (BLAST) available from the pion(R) system the labeled probe in the form of a hairpin National Center for Biotechnology Information. structure is linked to the primer. It is possible for the methods of the invention to be used in Quantitation in real time format may be on an absolute order to detect more than one gene of interest in the same basis, or it may be relative to a methylated DNA standard or 35 reaction. Through the use of several specific sets of primers, relative to an unmethylated DNA standard. The absolute copy amplification of several nucleic acid targets can be performed number of the methylated marker gene can be determined; or in the same reaction mixture. This may be termed “multiplex the methylation status may be determined by using the ratio ing'. Multiplexing can also be utilized in the context of between the signal of the marker under investigation and the detecting both the gene of interest and a reference gene in the signal of a reference gene with a known methylation (e.g. 40 same reaction. B-actin), or by using the ratio between the methylated marker The term “test sample” refers to biological material and the sum of the methylated and the non-methylated obtained from a mammalian Subject, preferably a human marker. Subject, and may be any tissue sample, body fluid, body fluid Real-Time PCR detects the accumulation of amplicondur precipitate, or lavage specimen. Test samples for diagnostic, ing the reaction, but alternatively end-point PCR fluorescence 45 prognostic, or personalized medicine uses can be obtained detection techniques may be used. Confirming the presence from cytological samples, from Surgical samples, such as of target DNA at the endpoint stage may indeed be sufficient biopsies, cervical conization or hysterectomy, from (formalin and it can use the same approaches as widely used for real fixed) paraffin embedded cervix or other organ tissues, from time PCR. frozen tumor tissue samples, from fresh tumor tissue samples, DNA methylation analysis has been performed success 50 from a fresh or frozen body fluid such as blood, serum, lymph, fully with a number of techniques which are also applicable in or from cervical scrapings, cervical Smears, cervical wash present methods of the invention. These include the MALDI ings and vaginal excretions. Such sources are not meant to be TOFF, Mass ARRAY (Ehrich, M. et al. 2005), Methylight exhaustive, but rather exemplary. A test sample obtainable (Trinh B. et al. 2001), Quantitative Analysis of Methylated from Such specimens or fluids includes detached tumor cells Alleles (Zeschnigk M. et al. 2004), Enzymatic Regional 55 and/or free nucleic acids that are released from dead or dam Methylation Assay (Galm et al., 2002), HeavyMethyl (Cot aged tumor cells. Nucleic acids include RNA, genomic DNA, trell, S E et al., 2004), QBSUPT, MS-SNuPE (Gonzalgo and mitochondrial DNA, single or double stranded, and protein Jones, 1997), MethylGuant (Thomassin H. et al. 2004), associated nucleic acids. Any nucleic acid specimen in puri Quantitative PCR sequencing, and Oligonucleotide-based fied or non-purified form obtained from such specimen cell microarray systems (Gitan R S et al., 2006). 60 can be utilized as the starting nucleic acid or acids. The test The number of genes whose modification is tested and/or samples may contain cancer cells or pre-cancer cells or detected can vary: one, two, three, four, five, six, seven, eight, nucleic acids from them. Preferably, the test sample contains nine or more genes according to Table 1 can be tested and/or squamous cell carcinomas cells or nucleic acids from Squa detected. Detection of epigenetic modification of at least one, mous cell carcinomas, adenocarcinoma cells or nucleic acids two, three, four, five, six, seven, eight, nine or more genes 65 of adenocarcinoma cells, adenosquamous carcinoma cells or according to Table 1 can be used as an indication of cancer or nucleic acids thereof. Samples may contain mixtures of dif pre-cancer or risk of developing cancer. The genes are pref ferent types and stages of cervical cancer cells. US 9,371,569 B2 13 14 Present invention also relates to Screening protocols for the In all aspects of the invention, the test sample is preferably screening of woman for cervical cancer and the precursors a cervical, cervicovaginal or vaginal sample of a woman. thereof. Traditionally the Pap Smear has been the primary The phrase “cervical cancer screening refers to organized screening method for the detection of abnormality of the periodic procedures performed on groups of people for the cervix, but its performance is suboptimal. Human Papilloma purpose of detecting cervical cancer. virus has been associated with the development of cervical The phrase “assaying for hr-HPV refers to testing for the cancer. Five high-risk types, 16, 18, 31, 45, and 58, and in presence of hr-HPV. There are various PCR based assays particular HPV types 16 and 18 account for approximately commercially available to measure hr-HPV copy number or 70% of all cervical carcinomas. A small percentage of women viral load in clinical samples. Many testing methods have showing persistent infection progress from Low-grade to 10 High-grade lesions. The introduction of methylation markers been used to detect the presence of HPV in cervicovaginal now adds a new dimension to the screening for and treatment specimens, including viral load quantification, Southern blot, of cervical lesions. Method for cervical cancer Screening may polymerase chain reaction (PCR), ViraPap (Life Technolo combine high-risk human papillomavirus (hr-HPV) testing gies, Gaithersburg, Md.), Hybrid Capture tube testing, and methylation testing; or cytological evaluation and methy 15 Hybrid Capture microtiter plate assays, and CISH. For lation testing; or hr-HPV testing and cytological evaluation instance, assaying for hr-HPV may be performed with the and methylation testing. FDA approved Hybrid Capture II assay (Digene Corp., Silver Thus, a further embodiment of the present invention relates Spring, Md.) with a probe cocktail for 13 carcinogenic types. to a method for cervical cancer detection or screening com The so-called “high risk” HPV types are those strains of prising the steps of: HPV more likely to lead to the development of cancer, while a) providing a test sample comprising cervical cells or nucleic “low-risk” viruses rarely develop into cancer. The list of acids from cervical cells; strains considered high risk is being adapted with the time and b) assaying the test sample of step a) for high-risk human the increase in epidemiological knowledge. As such, those papillomavirus (hr-HPV); hr-HPV types comprise, without being limited to, strains 16, c) if b) is positive for the presence of hr-HPV, assaying the 25 18, 31, 33,35, 39, 45, 51, 52,56,58, 59, 68, and 69. Preferred methylation status of at least one gene selected from the “high risk” HPV types are HPV 16 and HPV 18. group consisting of genes according to Table 1: The phrase “HPV 16 testing refers to testing for the pres d) if the gene of c) is methylated, refer the woman for colpos ence of hr-HPV type 16. Similarly, “HPV18 testing” refers to copy; testing for the presence of hr-HPV type 18. The various e) if the gene ofc) is unmethylated, refer the woman to a more 30 methods allowing type-specific HPV testing are well known regular screening for the presence of hr-HPV. to the person skilled in the art and are applicable in the The present invention relates further to a method for cer methods of present invention. For instance, testing for the Vical cancer detection or screening comprising the steps of: presence of hr-HPV-16 may be accomplished by PCR ampli a) providing a test sample comprising cervical cells or nucleic fication using primers specific for HPV type 16, which are acids from cervical cells; 35 known by the skilled in the art. b) assaying the test sample of step a) for hr-HPV: The phrase “performing cytological evaluation” refers to c) if b) is positive for the presence of hr-HPV, assaying the the cytomorphological assessment of cervical samples, methylation status of at least one gene selected from the which is usually performed by especially trained medical group consisting of genes according to Table 1, and/or staff. The various methods allowing cytological testing are typing the hr-HPV for the presence of HPV 16 and/or 40 well known to the person skilled in the art and are applicable HPV 18; in the methods of present invention. Cytological evaluation d) if the gene of c) is methylated, and/or HPV 16 and/or may be performed with the known Papanicolaou (PAP) smear HPV 18 positive, refer the woman for colposcopy; test. Alternative means for cytological evaluation include liq e) if the gene ofc) is unmethylated, refer the woman to a more uid based cytology with for example the ThinPrep technique regular screening for the presence of hr-HPV. 45 (Cytyc Corporation, Marlborough, Mass., USA). In a related embodiment, the invention provides for a The term “triaging refers to sorting out or classifying method for cervical cancer detection or screening comprising patients in order to establish priority of treatment's necessity, the steps of: priority of proper place of treatment, or any other priority in a) performing cytology evaluation on a test sample compris terms of patient management. ing cervical cells or nucleic acids from cervical cells; 50 The test sample will most of the time be obtained from a b) if a) is positive, assaying the methylation status of at least Subject Suspected of being tumorigenic or from a subject one gene selected from the group consisting of genes undergoing routine examination and not necessarily being according to Table 1: Suspected of having a disease. Alternatively the sample is c) if the at least one gene ofb) is methylated, refer the woman obtained from a subject undergoing treatment, or from for colposcopy; 55 patients being checked for recurrence of disease. d) if the at least one gene of b) is unmethylated, refer the Testing can be performed diagnostically or in conjunction woman to cytology testing on a more regular basis. with a therapeutic regimen. Testing can be used to monitor In a related embodiment, the invention provides for a efficacy of a therapeutic regimen, whether a chemotherapeu method for cervical cancer detection or screening comprising tic agent or a biological agent, such as a polynucleotide. the steps of: 60 Epigenetic loss of function of at least one gene selected from a) assaying the methylation status of at least one gene selected the group consisting of genes according to Table 1 can be from the group consisting of genes according to Table 1: rescued by the use of DNA demethylating agents and/or DNA b) if the at least one gene ofb) is methylated, perform cytol methyltransferase inhibitors. Testing can also be used to ogy testing: determine what therapeutic or preventive regimen to employ c) ifb) is tested positive, refer the woman for colposcopy; 65 on a patient. Moreover, testing can be used to stratify patients d) ifb) is negative, refer the woman to methylation testing on into groups for testing agents and determining their efficacy a more regular basis. on various groups of patients. US 9,371,569 B2 15 16 Demethylating agents can be contacted with cells in vitro Typically the kit will contain both a forward and a reverse or in Vivo for the purpose of restoring normal gene expression primer for a single gene or marker. If there is a Sufficient to the cell. Suitable demethylating agents include, but are not region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, limited to 5-aza-2'-deoxycytidine, 5-aza-cytidine, Zebu then the primer may also contain additional nucleotide resi larine, procaine, and L-ethionine. This reaction may be used dues that do not interfere with hybridization but may be useful for diagnosis, for determining predisposition, and for deter for other manipulations. Exemplary of Such other residues mining Suitable therapeutic regimes. Accordingly, the inven may be sites for restriction endonuclease cleavage, for ligand tion also provides for a method for predicting the likelihood binding or for factor binding or linkers or repeats. The oligo of Successful treatment or resistance to treatment of cancer nucleotide primers may or may not be such that they are with such agent. If the gene is methylated, the likelihood of 10 specific for modified methylated residues. The kit may Successful treatment is higher than if the gene is unmethy optionally contain oligonucleotide probes. The probes may lated, or methylated to a lesser degree. Conversely, if the gene be specific for sequences containing modified methylated is unmethylated, or methylated to a lesser degree, the likeli residues or for sequences containing non-methylated resi hood of resistance to treatment is higher than if the gene is 15 dues. The kit may optionally contain reagents for modifying methylated. methylated cytosine residues. The kit may also contain com In a related aspect, epigenetic loss of gene function(s) can ponents for performing amplification, such as a DNA poly identify the stage of the disease and from that the need of merase and deoxyribonucleotides. Means of detection may treatment. Accordingly, the invention provides for a method also be provided in the kit, including detectable labels on for predicting suitable treatment comprising determining the primers or probes. Kits may also contain reagents for detect methylation status of a gene or a combination of genes. If the ing gene expression for one of the markers of the present gene is methylated, the need of cervical resection is identi invention. Such reagents may include probes, primers, or fied; if the gene is unmethylated or methylated to a lesser antibodies, for example. In the case of enzymes or ligands, degree, it is decided that there is no need for cervical resec Substrates or binding partners may be sued to assess the tion. In cases of early stage (CIN) and carcinoma in situ, 25 presence of the marker. Kits may contain 1, 2, 3, 4, or more of abnormal tissue is removed by cryoSurgery, laser Surgery, the primers or primer pairs of the invention. Kits that contain conization, or simple hysterectomy (removal of the uterus). probes may have them as separate molecules or covalently Invasive cervical cancer is treated with radical hysterectomy linked to a primer for amplifying the region to which the (removal of the uterus, fallopian tubes, ovaries, adjacent probes hybridize. Other useful tools for performing the meth lymph nodes, and part of the Vagina). 30 ods of the invention or associated testing, therapy, or calibra To attain high rates of tumor detection, it may be necessary tion may also be included in the kits, including buffers, to combine the methods of the invention with established enzymes, gels, plates, detectable labels, vessels, etc. methods and/or markers for cervical cancer identification According to a further aspect, the invention also employs (Malinowski D, 2007), such as morphology-based detection or relies upon or utilizes oligonucleotide primers and/or methods, HPV methylation testing (Badal et al. 2004, Kalan 35 probes to determine the methylation status of at least one gene tarietal. 2004), KRAS and BRAF mutation detection (Kang selected from a group of genes consisting of JAM3. LMX1A, et al. 2007), chromosomal amplification (Rao et al. 2004), CDO1, NID2, ALX3, ALX4, AR, ARID4A, ATM, AURKA, protein expression (Keating et al. 2001) and HPV detection B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf248, methods (Brink et al. 2007): several HPV detection kits are C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, known in the art and commercially available, for example kits 40 CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, such as DigeneRHPV Test (Qiagen), AMPLICOR HPV Test COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, (Roche), HPV High-Risk Molecular Assay (Third Wave DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, Technologies), LINEAR ARRAY HPV Genotyping Test GADD45A, GATA4, GDAP1L1, GNB4, GPNMB, GREM1, (Roche), INNO-LiPAHPV Genotyping (Innogenetics), Pap Gst-Pi, HHIP, HIN1, HOOK2, HOXA1, HOXA11, HOXA7, illoCheck (Greiner Bio-One GmbH), PreTect HPV-Proofer 45 HOXD1, IGSF4, ISYNA1, JPH3, KNDC1, KRAS, LAMA1, (Norchip), NucliSENS EasyO HPV (BioMerieux), F-HPV LOC285016, LOX, LTB4R, MAL, MTAP, MYO18B, TypingTM (molGENTIX, S.L.) may be utilized. Such NDRG2, NOL4, NPTX1, NPTX2, OGFOD2, PAK3, PAX1, examples are not meant to be exhaustive, but rather exem PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, plary. RARA, RASSF1A, RBP4. RECK, RPRM, SALL4, Another aspect of the invention is a kit for assessing methy 50 SEMA3F, SLC5A8, SLIT1, SLIT2, SLIT3, SMPD1, lation in a test sample. Kits according to the present invention SOCS1, SOX1, SOX17, SPARC, SPN, SST, TAC1, TERT, are assemblages of reagents for testing methylation. They are TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, typically in a package which contains all elements, optionally WIF1, WIT1, WT1, XRCC3, and ZGPAT. Preferred probes including instructions. The package may be divided so that and their sequences bind to at least one of the polynucleotide components are not mixed until desired. Components may be 55 sequences listed in Table 2, FIG. 5B or to the complement in different physical states. For example, some components sequence thereof. Preferred primers and probes are selected may be lyophilized and some in aqueous solution. Some may from the primers and probes comprising or consisting essen be frozen. Individual components may be separately pack tially of the nucleotide sequences set forth in Table 1. Related aged within the kit. The kit may contain reagents, as described to this, the invention also provides for an isolated polynucle above for differentially modifying methylated and non-me 60 otide which consists of a nucleotide sequence listed in Table thylated cytosine residues. 1, Table 2 and FIG. 5B. Desirably the kit will contain oligonucleotide primers The above disclosure generally describes the present which specifically hybridize to regions within about 10 kbp. invention. All references disclosed herein are expressly incor within about 5 kbp, within about 3 kbp, within about 1 kbp. porated by reference. A more complete understanding can be within about 750 bp, within about 500 bp, within 200 bp or 65 obtained by reference to the following specific examples within 100 bp kb of the transcription start sites of the genes/ which are provided herein for purposes of illustration only, markers listed in Table 1. and are not intended to limit the scope of the invention.

US 9,371,569 B2 47 48 EXAMPLES Cell lines were treated for 3 days with low to high dose (200 nM. 1 uM or 5 uM) 5-aza-2'deoxycytidine (DAC), 200 nM Example 1 DAC with 300 nM trichostatin A (TSA) after 48 hours, or left untreated. Cells were split to low density 24 hours before Discovery of Methylation Markers in Cervical treatment. Every 24 hours DAC was refreshed. After 72 hours Cancer, Using Relaxation Ranking cells were collected for RNA isolation. RNA and DNA Isolation: To identify genes that are downregulated in cervical cancer From the frozen biopsies, four 10-um-thick sections were due to promoter hypermethylation and to enrich for those cut and used for standard RNA and DNA isolation. After genes that are most frequently involved in cervical cancer, a 10 cutting, a 3-um-thick section was stained with haematoxylin/ multistep approach was used combining eosin for histological examination and only tissues with Affymetrix expression microarray analysis on a panel of >80% tumor cells were included. Macrodissection was per frozen tissue samples from 39 human primary cervical formed to enrich for epithelial cells in all normal cervices. cancers to identify cancer-specific down-regulated For DNA isolation, cells and tissue sections were dissolved genes. 15 in lysis buffer and incubated overnight at 55° C. DNA was Affymetrix expression microarray analysis on a panel of 4 extracted using standard salt-chloroform extraction and etha different cervical cancer cell lines in which the expres nol precipitation for high molecular DNA and dissolved in sion of (hyper)methylated genes was re-activated upon 250 ulTE-4 buffer (10 mM Tris; 1 mM EDTA (pH 8.0)). For treatment with 5-aza-2'deoxycytidine (DAC) (blocking quality control, genomic DNA was amplified in a multiplex DNA methylation), and/or trichostatin A (TSA) (inhib PCR containing a control gene primer set resulting in prod iting histone deacetylase HDAC). ucts of 100, 200, 300, 400 and 600 bp according to the Data from both approaches were combined, and a novel BIOMED-2 protocol (van Dongen et al., 2003). non-parametrical ranking and selection method was applied RNA was isolated with TRIZol reagent (Invitrogen, Breda, to identify and rank candidate genes. Using in silico promoter The Netherlands) according to manufacturer's protocol. analysis we restricted the analysis to those candidate genes 25 RNA was treated with DNAse and purified using the RNeasy that carry CpG-islands. The new approach resulted in a sig mini-kit (Qiagen, Westburg, Leusden, The Netherlands). The nificant enrichment of hypermethylated genes: we compared quality and quantity of the RNA was determined by Agilent the first 3000 high-ranking candidate probes with lists of Lab-on-Chip analysis (ServiceXS, Leiden, The Netherlands, imprinted genes, X- located genes and known www.serviceXS.com). methylation markers. In addition, we determined the hyper 30 Expression Data: methylation status of the 10 highest ranking candidate genes Gene expression for 39 primary cancers and 20 cell line in both cervical cancers and normal cervices using COBRA samples was performed using the Affymetrix HGU 133 Plus (COmbined Bisulfite Restriction Analysis). 2.0 array with 54,675 probes for analysis of over 47,000 Material and Methods human transcripts. The labeling of the RNA, the quality con Primary Cervical Tissue Samples: 35 trol, the microarray hybridization and scanning were per For the expression microarray analysis, tissues from 39 formed by ServiceXS according to Affymetrix standards. For early stage frozen cervical cancer Samples were used from a labeling, ten microgram of total RNA was amplified by in collection of primary tumors Surgically removed between vitro transcription using T7 RNA polymerase. 1993 and 2003 (University Medical Center Groningen, Quality of the microarray data was checked using histo Groningen, The Netherlands). All cervical cancer patients 40 grams, boxplots and a RNA degradation plot. One cell line underwent gynecological examination for staging in accor sample was omitted because of poor quality. Using BioCon dance with the International Federation of Gynecology and ductor (Gentleman et al., 2004), present (P), absent (A) or Obstetrics (FIGO) criteria (Finan et al., 1996). Tumor marginal (M) calls were determined with the MASS algo samples were collected after surgery and stored at -80° C. rithm. MASS uses a non-parametric statistical test (Wilcoxon The stage of cervical cancer patients included 33 FIGO stage 45 signed rank test) that assesses whether significantly more 1B (85%) and 6FIGO stage IIA (15%). The medianage of the perfect matches show more hybridization signal than their cervical cancer patients was 46 years (IQ range 35-52 yr.). corresponding mismatches to produce the detection call for For COBRA and BSP (Bisulfite Sequencing PCR), 10 (of each probe set (Liu et al., 2002). The relaxation ranking the 39) primary cervical cancers and 5 controls (normal cer approach only relied on P-calls. Some samples were analyzed vix) were used. The age-matched normal cervical controls 50 in duplicate, and the profile of P-calls is highly similar (93 were women without a history of abnormal PAP smears or 95% of the probesets have an identical P/M/A call). any form of cancer and planned to undergo a hysterectomy for Relaxation Ranking Algorithm: benign reasons during the same period. Normal cervices were In order to identify the most promising markers that are collected after Surgery and histologically confirmed. methylated in cervical cancer, we assumed that Such markers Informed consent was obtained from all patients partici 55 should be silenced in cancer cells and upregulated upon re pating in this study. activation after DAC/TSA treatment; therefore, the best Cervical Cancer Cell Lines: methylation markers will be genes represented by probes Four cervical carcinoma cell lines were used: HeLa (cer with: vical adenocarcinoma, HPV 18), SiHa (cervical squamous no expression in primary cervical cancers: P-calls=0 out of cell carcinoma, HPV16), CSCC-7 (non-keratinizing large 60 39 cancers cell cervical squamous cell carcinoma, HPV 16) and CC-8 no expression in (untreated) cervical cancer cell lines: (cervical adenosquamous carcinoma, HPV45). HeLa and P-calls=0 out of 4 cell lines SiHa were obtained from the American Tissue Type Collec expression in cervical cancer cell lines treated with DAC tion. CSCC-7 and CC-8 (Koopman et al., 1999) were a kind (or DAC in combination with TSA): P-calls=15 out of 15 gift of Prof. G.J. Fleuren (Leiden University Medical Center, 65 treated cell lines Leiden, The Netherlands). All cell lines were cultured in To select for those gene probes that would be the best DMEM/Ham's F12 supplemented with 10% fetal calfserum. candidate hypermethylated genes in cervical cancer, we US 9,371,569 B2 49 50 present the relaxation ranking algorithm. Probe sets were iii. Zez (number of P-calls for probe in treated cell ranked, not primarily based on the number of P-calls and thus lines larger or equal compared to condition) explicitly setting thresholds, but primarily driven by the num d. under very strict conditions (x=0, y=0, Z=15) no probes ber of probe sets that would be picked up, based on selection were found, while under the most relaxed conditions criteria (the number of P-calls in primary cancers, untreated 5 and treated cell lines). The stricter (e.g. P-calls: 0-0-15) these (x=39, y=4, Z=0) all probes were returned. For all com selection criteria, the lower the number of probes that meet binations of x, y and Z, the number of probes that com with these criteria; while if the conditions become more and plied (w), was stored more relaxed (higher number of P-calls in primary cancers (2) The data was sorted with was primary criterion (ascend and untreated cell lines, and lower number of P-calls in ing), followed by X (ascending), y (ascending) and Z (de treated cell lines), the more probes will comply. In the end, scending) using P-calls: 39-4-0 as criteria, all probe sets were returned. (3) This sorted dataset was analyzed row per row. In row i, the This way, there was no need to define a prior threshold for w, probes retrieved with criteriax, y, z, were compared with the number of P-calls. the list of probes, already picked up in rows 1 to i-1. If a The following sorting method was applied: 15 probe did not occur in this list, it was added to the list (1) All possible conditions were generated and the number of (4) This process continued until there were m (user-defined) probes that were picked up under these conditions was probes in the list calculated: DNA Methylation Analysis Using COBRA and Bisulphate a. the number of samples with expression (P) of a certain Sequencing: probe in To validate the (hyper)methylated Status of candidate gene i. primary cervical cancer Samples is called X, probes, DNA extracted from 10 cervical cancers and 5 normal ii. cervical cancer cell lines is called y, cervices were analyzed using BSP and COBRA. Bisulfite iii. treated cervical cancer cell lines is called Zsample modification of genomic DNA was performed using the EZ b. all combinations of x, y and Z are made DNA methylation kit (Zymogen, BaseGlear, Leiden, The i. X (the number of P-calls in primary cancers) varies Netherlands). The 5' promoter region of the tested gene was from 0 to 39 25 amplified using bisulfate treated DNA. PCR primers for ii. y (the number of P-calls in untreated cell lines) from amplification of specific targets sequences are listed in Table O to 4 4. COBRA was performed directly on the BSP products as iii. Z (the number of P-calls in treated cell lines) from 0 described by Xiong et al. (Xiong and Laird, 1997) using to 15 30 digestions with BstUI, Taqland/or Hinflaccording the manu iv. In total, 3200 combinations of x, y and Z can be made facture's protocol (New England Biolabs Inc., Beverly, ... a probeset was found under each of these generated Mass.). For sequence analysis, the BSP products were puri conditions x, y and Z if: fied (Qiagen) and Subjected to direct sequencing (BaseGlear, i. X, sX (number of P-calls for probe in primary Leiden, The Netherlands). Leukocyte DNA collected from cancers Smaller or equal compared to condition) AND anonymous healthy volunteers and in vitro CpG methylated ii. yatsy (number of P-calls for probe in untreated 35 DNA with SssI (CpG) methyltransferase (New England cell lines Smaller or equal compared to condition) Biolabs Inc.) were used as negative and positive control, AND respectively. TABL E 4.

list of primers used for BSP (1. +1 is transcription start site (TSS); Several primer pairs were tested, however, none worked)

Start End .Name . Forward primer (5'-3') . Reverse primer (5'-3') . Ta position' position . RefSeq,

DAZL TTTGGGGGTGATGTGTGTGTTT TCTCCCTCAACT CACCATAATA 54 .-161. .312 NMOO1351

ADARB1? .NM O15834

SYCP3 AAAATTTAAAAATTGGAAGGTATT ACCTCACTAATCAAAAACAACCTCT 54 -2O8 ... --186 NM 1536.94 AGG

AUTS2 TTTTAAAAGTGATAAAGTTGGTTA . CCCTTTTCTTTCTCCTCTCTTTCT 56 ... --3 OO ... -184 NMO15570 TGG. T.

INNAT GGTTAGGGATTGGGGAGAA GCTAAACTTACCTACAACAACAC 54 -271 .21O NMOO5386

SST GGGGTATGTGGAATTGTGTG AAA TCT CCT TAC CTA CTT CCC C 54 -185 +276 NMOO1 O 48

HTRA3 GTYGGTTTTGTYGTTATGTAGGY AAC TTC ACT TCC, TCC CTA ACC st ... -- 19 O ... -- 6 22 NMO53O44

ZFP42 AGTAGGTGTTTGTTGAAGATAG ACT CAT AAC ACA CAT AAC CAT C 60 +3O8 +58O NM 1749 OO

NPTX1 GGTAGTGGGGGTTTGATAG AAATAATCTCCTTCTACTACAACAC 54 -2 ... +372 NMOO2522

GDA TATAGAAGGTGGAGGAAGTTGA CACCTCCATAAAACAAATCCAAA 54 -239 ... --194 NMOO 4293

CCNA1 TATAGTTGGAGTTGGAGGGT AAACAACTAACAAATACACTAAAA 54 -279 . .146 NM 1536.94 US 9,371,569 B2 51 52 Results BSP was used to amplify the CpG-islands of these candi To identify novel markers that are methylated in cervical date genes using bisulfite-treated DNA and COBRA to deter cancer, we applied a multistep approach that combines re mine the methylation status. CCNA1 (at position 49) was expression of silenced hypermethylated genes in cervical included as a positive control for the highest listed, reported cancer cell lines (using DAC and DAC/TSA), downregulated 5 cervical cancer specific methylation gene promoter. BSP/ expression in 39 cervical cancers expression, and selection of COBRA of CCNA1 revealed that 6 of 10 carcinomas are candidate markers using a relaxing ranking algorithm. The methylated at the restriction enzyme sites (T1, T3, T5, T7. T9 best profile of a candidate marker would be: no expression in and T10 in FIG. 3). Sequence analysis of the BSP-products any of the 39 cervical primary cancers and 4 untreated cancer (on average 7-9 independent clones for each carcinoma) of cell lines, but re-activation of expression after demethylation 10 these 10 carcinomas revealed that in 6 carcinomas the pro and/or blocking of histone deacetylation in all 15 cell lines moter is hypermethylated in good agreement with the treated with various combinations of DAC/TSA (P-calls: 0-0- COBRA results (FIG. 3C). 15). However, none of the probe sets showed this ideal profile. To generate a list of candidate genes, a relaxation ranking TABLE 5 algorithm was applied. 15 Methylation status using COBRA of the 10 highest ranking The only variable used in the relaxation ranking is the gene promoters. Gene selected for further validation after number of probes we would like to retrieve. As shown in FIG. applying additional criteria. Included is CCNA1 on position 1, the number of probes retrieved (w) with parameters x,y and 47 (original position 241) as the highest ranking cervical cancer-associated hypermethylated gene. Methylation Z (the number of P-calls in respectively primary tumor status was determined by BSPCOBRA samples, untreated and treated cell lines) follows a complex See FIG.3 and FIG. 4). profile which consists not only of additive elements, but also interactions between the parameters. In general, the number Gene Chromosomal Methylation Methylation of P-calls in primary cancer samples (X) has the largest influ Rank symbol location in cancer in normal ence on w. The sorting methodology has the advantage that no 25 1 DAZL 3p24.3 9.9 5/5 2 ADARB1 21q22.3 Nd Nd cut-off values have to be chosen for x, y and Z, and therefore 3 SYCP3 12q 9.9 5/5 there is no need to implicitly link a relative weight factor to 4 AUTS2 7q11.22 O.9 OS the parameters. 5 NNAT 20a11.2 9.9 5/5 To calculate the most optimal number of potentially hyper 6 SST 3q28 7/9 OS methylated candidate markers for further analysis, we esti 30 7 HTRA3 4p16.1 1.9 OS mated this number based on known (i.e. described in litera 8 ZFP42 4q35.2 9.9 5/5 ture) methylation markers in cervical cancer. Forty-five 9 NPTX1 17q25.1 5,10 Of 5 known methylation markers were found using text-mining 10 GDA 9q21.13 O.9 OS using GeneCards (Rebhan et al., 1997) for aliases/symbols to 47 CCNA1 6.10 OS query PubMed through NCBI E-Utils. The position of the 35 markers after ranking (“observed') was determined as shown Table 5 summarizes the methylation status of the 10 high in the step plot in FIG. 2. If the markers would be randomly est ranking genes in 10 cervical cancer and 5 normal cervices distributed in the ranking, the profile would be similar to the using COBRA. One gene (ADARB1 at rank 2) could not be curve, marked expected. This expected curve is not a analyzed for methylation as no specific BSP products could straight line, but is calculated based on whether a probe could 40 be assigned with a gene symbol and taking probes into be amplified using several combinations of primer pairs. account that are associated with a gene that is already asso Interestingly, using the BSP products of the other 9 listed ciated with an earlier selected probe. The number of observed genes, 7 (78%) showed methylation in carcinomas (Table 5). methylation markers has in general the same slope as Four genes are hypermethylated in all 9 tested cancers, while expected. However, until about 3000 probes, the slope of the 45 for SST (7 of 9 carcinomas), HTRA3 (1 of 9 carcinomas) and number observed markers versus the number of selected NPTX1 (5 of 10 carcinomas) not all tested carcinomas are probes (in dashed lines) cannot be explained if the markers hypermethylated. FIG. 4 shows representative methylation would be randomly distributed as its steepness is much analysis of 3 genes using COBRA. Three (NNAT, SST and higher. When selecting more than 3000 probes, the slope NPTX1) of the 7 hypermethylated gene promoters have been Suddenly decreases to a level that is close to random distri 50 reported to be methylated in tumors previously. Taken these bution. This enrichment can also statistically be proven. data together, these findings showed that the relaxation rank Therefore, we selected the first 3000 probes, referred to as ing algorithm resulted in a very significant enrichment for TOP3000, in the ranking for further analysis. In this genes with a positive methylation status. TOP3000 list, 2135 probes are associated with a gene symbol, A cervical-cancer-specific hypermethylated marker is only of which 1904 are unique. 55 Validation of the 10 Highest-Ranking Candidate Genes Using of relevance for the diagnosis of (pre-) malignant disease in COBRA: case normal cervical epithelium is not methylated. COBRA In order to validate whether the highest ranking genes analysis of 5 normal cervices for all 9 genes revealed that 4 represent markers that are functionally hypermethylated in genes (DAZL, SYCP3, ZFP42 and NNAT) are hypermethy cervical cancer, we performed COBRA on bisulfite-treated 60 lated in all 5 samples (Table 5). On the other hand, of the 7 DNA of 10 cervical cancers and 5 normal cervices. For this genes hypermethylated in cervical cancer specimens, 3 genes analysis we focused on those first 10 genes from the highest (SST, HTRA3 and NPTX1) did not show DNA methylation ranking probe-list (Table 5) that: in any of the normal cervices of 5 independent individuals. represent a known gene (i.e. gene symbol) We observed the same methylation profile for CCNA1 that contain a CpG-island surrounding the TSS 65 was reported previously as a cervical cancer specific gene are located on any chromosome except chromosome X (Kitkumthornet al., 2006) with hypermethylation in only 6 of are expressed in less than 15 carcinomas 10 tumors but none of the 5 normals (Table 5: FIG. 3). US 9,371,569 B2 53 54 Example 2 Material and Methods Samples: BROAD Analysis: Genome-Wide Promoter A total of 201 frozen tissue samples (87 cervical cancer Alignment samples, the majority derived from squamous cell carcino mas; and 114 normal tissues) were collected by UMC The “Database of Transcription Start Sites” (DBTSS) (Su Groningen. If the tissue contained more than 20% stromal Zuki et al., 2004) mapped each transcript sequence on the cells, the samples were macro-dissected to enrich for tumor human draft genome sequence to identify its transcriptional cells. start site, providing more detailed information on distribution DNA Isolation and Bisulphite Modification: 10 DNA was isolated using proteinase K digestion and phe patterns of transcriptional start sites and adjacent regulatory nol/chloroform extraction. DNA concentration was measured regions. The promoters of the above identified TOP3000 using NanoDrop Spectrophotometer. From each sample, up genes were separately mapped on the genome-wide align to 2 g of genomic DNA was converted using a bisulphite ment of all promoter associated CpG islands. All the promoter based protocol (EZ DNA Methylation KitTM, ZYMO sequences were Subsequently aligned by clustalW algorithm 15 Research, Orange, Calif.). (Li 2003; Thompson et al., 1994). Treeillustrator (Trooskens Detection of Hypermethylation: et al., 2005) was used to visualize the large guide tree in Methylation specific PCR (MSP) primers were designed addition to indicating the location of the known markers. for each of the genes assessed for (hyper)methylation. An Some regions on the “circle” are denser in known markers example on primer design spanning a large region of the than others, indicating that there might be a sequence mecha promoter is provided in FIGS.5A and 5B for ALX4. nism located in the small region around the TSS which makes For some genes more primer pairs were designed giving a certain genes more methylation-prone. The genes were total of 424 different assays. These assays were applied on 8 selected as candidates to be methylated if they were located in sub-arrays of 2 OpenArrayTM plates by BioTrove Inc. The a cluster, i.e. less than 9 nodes (distance to the closest neigh beta-actin assay was applied on each Sub-array as an internal boring marker) away from a marker already described in the 25 control. Quality control was performed using an in vitro literature. These genes were assigned a score, calculated as methylated DNA sample and a negative control sample. The follows: if the gene is a known literature marker, score +10, if selectivity and the reproducibility were checked. After DNA a known marker is one node away, score +9, if there are conversion and purification, beta-actin copy number was markers two nodes away: addition to score number of mark determined by qMSP. The equivalent of 1500 beta-actin cop 30 ies per sample was applied per sub-array of an OpenArrayTM ers*8, etc. The genes were ranked according to this score. plate on a real-time qPCR system (BioTrove Inc.) using the A final gene selection was made based on the ranking, the DNA double strand-specific dye SYBRgreen for signal opportunity to design primers, genes to be known as tumor detection. Suppressor genes and expert knowledge on their function, The cycling conditions were: 90° C.-10 seconds, (43°C. 18 history and mutation status in other cancertypes. Also known 35 seconds, 49° C. 60 seconds, 77° C. 22 seconds, 72° C. 70 genes from literature and previous research were included for seconds, 95° C. 28 seconds) for 40 cycles, 70° C. for 200 confirmation. seconds, 45° C. for 5 seconds. A melting curve was generated A final selection of markers resulting from the above set in a temperature range between 45° C. and 94° C. out approaches, were tested on tissue using the Base5 methy Analysis of Methylation: lation profiling platform (Straub et al. 2007). Differential 40 For each combination of assays and samples two param methylation of the particular genes was assessed using Base5 eters were collected using an algorithm which is part of the methylation profiling platform as follows: DNA was standard data analysis package offered by the Supplier. The extracted from cervical samples, bisulfite converted, and parameters were the Ct value (threshold cycle number) of the selected regions of the particular genes were amplified using assessed amplicon and the melting temperature of the primers whose sequence represented converted or non-con 45 assessed amplicon. The following data analysis workflow verted DNA sequences. Amplification was monitored in real was applied to the results created by the software which came time set up using SYBRgreen. Data analyses designed to cope with the system OpenArrayTM system: Data was collected for with inherent variance (i.e., noise) in measured Ct and Tm each combination of assays and samples in the two sets of values were applied to withhold 112 different assays for samples used. Results were filtered using the following detecting differential methylation of ALX3, ALX4, AR, 50 approach. Read outs from not loaded reaction spaces were removed from analysis. Technical Control assays were ARID4A, ATM, AURKA, B4GALT1, BMP2, BMP6, removed from the data set. Assays known to not work for BNIP3, C13orf18, C16orfA8, C90rf19, CALCA, CAMK4, other than biological reasons were removed from the analy CCNA1, CCND2, CDH1, CDH4, CDK6, CDKN1B, sis. Per Sub-array, signals were only interpreted if there was a CDKN2B, CLSTN2, CLU, COL1A1, CPT1C, CTDSPL, 55 positive beta-Actin call. Ct values >0 for each gene were CYCLIND2, DAPK1, DBC1, DDX19B, DKK2, EGFR, normalized using the Ct values collected for the gene beta EGR4, EPB41L3, FOS, FOXE1, GADD45A, GATA4, Actin. This resulted in two files containing the results for each GDAP1L1, GNB4, Gst-Pi, HHIP, HOOK2, HOXA1, set of sample. 201 samples were tested of which 6 gave HOXA11, HOXA7, IGSF4, ISYNA1, JAM3.JPH3, KNDC1, invalid results. In total 79,170 reactions were performed of KRAS, LMX1A, LOC285016, LOX, MTAP, MYO18B, 60 which 74,110 were valid. For the data analysis, 2 boundaries NOL4, NPTX1, OGFOD2, PAK3, PAX1, PDCD4, were defined: an upper bound on beta-Actin-normalized-Ct PHACTR3, POMC, PRKCE, RAD23B, RALY, RARA, (banct) and a lower bound on Melting Temperature (Tm). RBP4, RECK, RPRM, SEMA3F, SLC5A8, SLIT1, SLIT2, Samples below the banct boundary and above the Tm bound SLIT3, SMPD1, SOCS1, SOX1, SPARC, SPN, SST, TERT, ary are considered to be “methylated, others (including all TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, UGT1A1, 65 samples with no signal, i.e. Ct.40) are classified as “unm WIF1, WIT1, WT1, XRCC3, and ZGPAT in cervical cancer ethylated. In both dimensions the set of candidate bound tissue samples. aries consists of all values in between 2 measurements, plus US 9,371,569 B2 55 56 infinity (the equivalent of no boundary). The set of candidate The sample set selected for the Lightcycler analysis was models for “methylated then consists of all combinations of also previously used in the Base 5 analysis in order to make a candidate Tm lower bound and a banct upperbound. A score compared analysis: a total of 27 cervical tumor samples and 20 controls (frozen tissue) were collected by UMC Gronin is computed for each of these candidate models, as follows. gen. Count: cancers inside boundaries=true positives (TP), can 5 cers outside boundaries=false negatives (FN), normals inside boundaries=false positives (FP), normals outside TABLE 6 boundaries=true negatives (TN). A binomial test was applied The 64 selected assays which were applied on the Lightcycler platform to find out how unusual it is to have at least TP Successes in (TP+FP) trials where the probability of success is (TP+FN). 10 No Assays Base 5 ranking The lower this probability value is the better. Then quality 1 LMX1A 9513 1 control data were taken into account to determine the most 2 SLIT2. 23681 2 robust boundaries. Using the standard deviations (StDevOC) 3 ISYNA1 19726 3 4 EPB41L3 19071 4 observed in the QC, a series of increasingly “noisy” datasets 5 WT1 1 5 were generated. The measurements are replaced by a value 15 6 DKK2 23973 6 randomly selected from a normal distribution with average 7 ALX3 2518O 7 equal to the observed measurement and standard deviation 8 AM3 8 equal to StDevOC multiplied by a value that gradually (10 9 JPH3 12611 9 10 SLIT2. 23672 10 noise levels) increases from 0 to 2. Each time the score of the 11 SOX1 27153 11 candidate model is computed by applying the 2 steps above 12 SOX1 271.59 12 (i.e., count and binomial test). All these scores (11 in total: 1 13 RALY 19607 13 14 RPRM 2 14 for “no noise' and 10 for noise levels 0.2,0.4, . . . . 2) are 15 CDH4 24735 15 added up to obtain the ultimate accumulated score. The can 16 CPT1C 23912 16 didate model with the best (i.e. lowest) accumulated score is 17 SLIT2. 23676 17 retained. This same score of the best candidate model for each 25 18 PAX1 27211 18 marker is also used for ranking the markers. 19 DKK2 23970 19 Results 2O TERT 23702 2O 21 NOL4 19645 21 A high throughput, real-time methylation specific detec 22 HOXA11 23844 22 tion platform was applied on two groups of samples isolated 23 CALCA 2 23 from cervical cancer tissue and from corresponding normal 30 24 C13orf18 19885 24 cervical tissue. In this study it was shown that a number of 25 PAX1 27210 25 26 WIT1 24567 26 genes are differentially methylated in cervical cancer. We 27 GATA4 13295 27 identified 112 different assays for detecting 96 different genes 28 SLIT1 23651 28 being differentially methylated in human cervical cancertis 29 LOC285O16 22940 29 Sue and normal cervical tissue control samples. The genes 30 POMC 30 identified are ALX3, ALX4, AR, ARID4A, ATM, AURKA, 35 31 Gst-Pi New3 32 32 DAPK1 34 B4GALT1, BMP2, BMP6, BNIP3, C13orf18, C16orf248, 33 GDAP1L1 19773 35 C9orf19, CALCA, CAMK4, CCNA1, CCND2, CDH1, 34 TFPI-2 36 CDH4, CDK6, CDKN1B, CDKN2B, CLSTN2, CLU, 35 TWIST1 9329 37 COL1A1, CPT1C, CTDSPL, CYCLIND2, DAPK1, DBC1, 36 SST 23808 38 DDX19B, DKK2, EGFR, EGR4, EPB41L3, FOS, FOXE1, 40 37 EGR4 24277 39 GADD45A, GATA4, GDAP1L1, GNB4, Gst-Pi, HHIP, 38 C16orf248 22922 45 39 DBC1 23879 46 HOOK2, HOXA1, HOXA11, HOXA7, IGSF4, ISYNA1, 40 GDAP1L1 19775 47 JAM3, JPH3, KNDC1, KRAS, LMX1A, LOC285016, LOX, 41 OGFOD2. 23131 48 MTAP, MYO18B, NOL4, NPTX1, OGFOD2, PAK3, PAX1, 42 ALX4 25062 49 PDCD4, PHACTR3, POMC, PRKCE, RAD23B, RALY, 45 43 TLL1 24051 51 RARA, RBP4. RECK, RPRM, SEMA3F, SLC5A8, SLIT1, 44 CTDSPL 23795 52 45 CYCLIND21 58 SLIT2, SLIT3, SMPD1, SOCS1, SOX1, SPARC, SPN, SST, 46 COL1A1 23253 65 TERT, TFPI-2, TLL1, TNFAIP1, TRMT1, TWIST1, 47 CDK6 9703 71 UGT1A1, WIF1, WIT1, WT1, XRCC3, and ZGPAT 48 CDH1 17968 76 The resulting assays have the assay details provided in 50 49 SOCS1 2.3595 78 Table 1, Table 2, and FIG. 5B. 50 FOXE1 13314 91 51 BMP2 17901 94 Example 3 52 AURKA 24802 110 53 SEMA3F 23485 120 S4 PAK33 121 Further Assay Selection: Base 5 Lightcycler 55 HOXA72 125 Platform 55 56 CTDSPL 23804 127 57 NPTX1 2 136 58 SLIT1 23653 164 Of the different assays listed in Table 1 previously identi 59 SMPD1 24061 174 fied using the Base5 methylation platform, the top 63 ranked 60 GADD45A 24463 250 assays plus ?-actin (ACTB) were transferred to the Lightcy 61 KRAS 24235 281 cler platform in order to further fine-tune the selection of the 60 62 RECK 18940 321 best cervical cancer methylation markers. This platform 63 UGT1A1 22912 341 allows the assessment of markers in a system which is closer 64 Beta Actin Internal control to, and provides information valuable for the Subsequent development of a final, scaled up MSP assay. The 64 assays Tissue slides were deparaffinized using 100% xylene fol (Table 6) were applied on a 384 well plate by Sigma. Six 65 lowed by 100% ethanol. Pellet was resuspended in a buffer repeats of the assay set fitted on a 384 well plate. The samples containing SDS-proteinase K, and DNA was extracted with were randomized per plate. phenol-chloroform followed by ethanol precipitation. DNA US 9,371,569 B2 57 58 concentration was measured using NanoDrop Spectropho All these results were taken into account to decide which tometer. From each sample, up to 3 jug of genomic DNA was assays should be further verified on cervical tissue samples converted using a bisulphite based protocol (EZDNA Methy collected by Ulg (normal PE tissue samples #13, cancer PE lation KitTM, ZYMO Research). After DNA conversion and tissue samples #17) and/or UMCG (normal frozen tissue purification, equivalent of 20 ng of g|NA was used per reac samples #20, cancer frozen tissue samples #27). tion. All the samples were tested on Lightcycler using Syber Seventeen (CCNA1, CDO1, 55928, CDO1, 55929, green as detector and the amplicon size was determined by GREM1, GPNMB, HIN1, HOXD1, LAMA1, LTE4R, MAL, capillary electrophoresis. NDRG2, NID2, NPTX2, RASSF1A, SALL4, SOX17, and Quality control was performed using in vitro methylated TAC1) additional good performing assays were also selected DNA sample, unmethylated DNA sample (Chemicon Inter 10 for further verification on the cervix tissue samples. These national, CA, USA; Cat.#57821 and Cat.#57822) and no candidates were taken from other in-house cancer projects, template control sample (H2O). From the Lightcycler plat and were not tested on the Biotrove/Lightcycler platform as form, the Ct values (cycle number at which the amplification described above. curves cross the threshold value, set automatically by the DNA was isolated from the cervix tissue samples using a Software) and melting curves (Tm) were generated. From the 15 phenol-chloroform procedure, quantified using the picogreen capillary electrophoresis platform, size of the amplicon and method and 1.5ug of DNA was bisulphite treated using the intensity of the signal detected were generated. For each ZYMO kit. assay, Tm and amplicon size parameters were determined in qMSPs were carried out in a total volume of 12 Jul in 384 in vitro methylated DNA sample, unmethylated DNA sample well plates in an ABI PRISM 7900HT instrument (Applied and no template control sample. The measured Tm and ampli Biosystems). The final reaction mixture consisted of in-house consize values were compared to the calculated values. If the qMSP buffer (including 80.4 nmol of MgCl2), 60 nmol of Tm or amplicon size values were out of the range of the each dNTP, 0.5 U of JumpStart Taq polymerase (SIGMA), 72 calculated ones, the assay was considered as non specific and ng of forward primer, 216 ng of reverse primer, 1.92 pmol of disqualified. All the 64 assays were specific. Molecular Beacon detection probe, 6.0 pmol of ROX (passive A sample is considered methylated if Ct is under 40 and if 25 reference dye) and 72 ng of bisulphite converted genomic Tm and amplicon size are within the boundaries of Tm+/-2 DNA. Thermal cycling was initiated with an incubation step degrees and amplicon size--/-10 bp. The intensity of the band of 5 minutes at 95°C., followed by 45 cycles (95°C. for 30 detected by capillary electrophoresis had to be higher than 20. seconds, 57°C. for 30 seconds, 72° C. for 30 seconds). A Those evaluation criteria have been developed based on con finalizing step was performed at 72° C. for 5 minutes to cordance with existing Molecular Beacon based qMSP 30 conclude cycling. These conditions were similar for all the assays, to ensure that the conclusions drawn from these data test genes as well as for ACTB. Cell lines in vitro methylated would be predictive of MSP assays developed subsequently. DNA sample and unmethylated DNA sample (Chemicon DNA methylation calls were compared between cervical International, CA, USA; Cat.#57821 and Cat.# S7822) were cancer and control patients. An assay ranking with the set of included in each run as positive and negative controls, and samples was generated and the results are Summarized in the entered the procedure at the DNA extraction step. Primers and methylation table of FIG. 6. A one-tailed Fisher's exact test 35 molecular beaconsequences used for the different qMSPs are was used as a scoring function to rank the candidate markers. summarized in Table 1 and Table 3. Corresponding amplicons The calculation of Fisher's exact test was based on a formula are summarized in Table 2. as described by Haseeb Ahmad Khan in “A visual basic Ct values were determined using the SDS software (ver software for computing Fisher's exact probability” (Journal sion 2.2.2.) Supplied by Applied Biosystems with automatic of Statistical Software, vol. 08, issue i21, 2003). 40 baseline settings and threshold. The slopes and R values for A comparison between the results coming from the Base 5 the different standard curves were determined after exporting (Biotrove) and the Lightcyclerplatforms has been performed. data into MS Excel. Most of the interesting assays discovered on the Base 5 plat As an example, FIG. 7 shows the amplification plot form were confirmed on the Lightcycler platform. obtained for the standard curve for TAC1 56.187 (960000 45 copies to 9.6 copies of the gene) and FIG. 8 shows the ampli Example 4 fication plot obtained for the standard curve and for all samples for TAC1 56187. The Ct values plotted against the Log Copies of TAC1 56.187 (FIG.9) give a R of 0.9995 and QMSP the efficiency of the reaction is 99.35%. In addition to the test genes, the independent reference Seventeen assays (ALX3, C13ORF18, DBC1, EPB41L3, 50 GATA4, HOXA11, JAM3, JPH3, LMX1A, NOL4, PAK3, gene f3-actin (ACTB) was also measured. The ratios between SLIT2. 23672, SLIT2. 23676, SOX1, TERT, TFPI2 and the test genes and ACTB were calculated to generate the test TWIST13) were further selected based on their performance result. The samples were classified as methylated, unmethy on the Biotrove and Lightcycler platforms and on comple lated, or invalid based on the decision tree shown in FIG. 10. mentarity analysis to maximize discriminatory power. For A provisional cut-off was defined for each gene, chosen these assays, qMSPs using Molecular Beacon as detection 55 based on the greater of either the highest value seen among system were designed (3 designs, if possible, were evaluated the controls or a value 3 times the standard deviation of the per assay) and tested on control samples. For this selection, values from control samples. assays were judged on several criteria, including background The one-tailed Fisher's exact test as described above was fluorescence, dynamic of the curve, and level of fluorescence used as a scoring function to rank the candidate markers generated. PCR material was used for generating standard 60 (Journal of Statistical Software, vol. 08, issue i21, 2003). curves for quantification of the results. Table 7 summarizes the results obtained for TAC156187. Five assays did not meet the desired specifications Table 8 summarizes the results obtained for all the tested (EPB41L3, SOX1, SLIT2. 23672, DBC1, and markers on tissue samples. The individual performances of SLIT2. 23676) and may be redesigned in a later phase or can the assays are shown in FIG. 11 and the assays are ranked be used on another detection platform. The remaining 12 65 according their p-value (Fisher's exact test). The best per assays were further tested on converted DNA of cervix cancer forming markers were further tested on clinical samples cell lines. (scrapings).

US 9,371,569 B2 65 66 Example 5 DNA was extracted from the scraped cells using standard Best Performing Markers Tested on Clinical Cervical salt-chloroform extraction and ethanol precipitation for high Scraping Samples molecular DNA, dissolved in 250 uL TE-4 buffer (10 mM Cervical scraping samples were collected under the Cervi Tris; 1 mM EDTA, pH 8.0) and kept at -20°C. until tested. cal Cancer Clinical Collaborative Research Agreement study Presence of high risk HPV was analyzed by PCR using of ONCO with the Gynecology Department of the UMCG hospital. The scraping samples were taken from patients who HPV 16 and HPV 18 specific primers on DNA of the scraping were referred to the hospital with an abnormal PAP smear or samples. On all HPV 16- or HPV 18-negative cases, general because they were suspected for cervical carcinoma. Gyne primer-mediated PCR was performed using two HPV con cological examination under general anesthesia was per sensus primer sets, CPI/CPIIG and GPS+/6+, with subse formed in all cervical cancer patients for staging in accor 10 quent nucleotide sequence analysis, as described previously dance with the International Federation of Gynecology and by Wisman etal Inti cancer 2006. Obstetrics (FIGO) criteria. Control scraping samples were qMSP was performed after bisulphite treatment on dena taken from women who visited the hospital for a non-malig tured genomic DNA. The assays were carried out as described nant condition, e.g. fibroids, prolaps uterior hypermenorrhea, above. The samples were classified as methylated, unmethy and who were scheduled to undergo a hysterectomy. While lated, or invalid as described above. The results obtained for the patient was under general anesthesia, the cervix was 15 all the tested markers on scraping samples from cervical scraped with an Ayres spatula and brush. The scraped cells cancer patients and from control patients were ranked accord were suspended in 5-ml PBS. Cytospins for cytomorphologi ing their p-value (Fisher's exact test) (Table 9). Some markers cal assessment were made (/S Volume). Cytospins were Papa have a higher sensitivity for squamous cell carcinoma than for nicolaou stained and routinely classified according to a modi fied Papanicolaou system (Hanselaar AG. Kwaliteit van adenocarcinoma (NID2, JPH3, CCNA1) and some markers cytopathologisch onderzoek in het herziene bevolking have a higher sensitivity for adenocarcinoma than for squa sonderzoek naar baarmoederhalskanker. Nederlands Tijd mous cell carcinoma (JAMS, CDO1, HOXA11). schrift voor Obstetrie en Gynaecologie 1996; 109:207-210) Various combinations of markers were evaluated to see if without knowledge of the clinical data. The remaining 4-ml of such a combination could increase the sensitivity while still the scraped cells was centrifuged, washed, aliquoted, Snap maintaining a high level of specificity. In all cases, if any frozen in liquid nitrogen and stored at -80° C. DNA was 25 marker of a combination panel was positive, the sample was extracted using standard salt-chloroform extraction and etha classified as methylated. Examples of the performance of nol precipitation. DNA of the pellet was used for qMSP of a combination of markers are summarized in Table 10. It can be panel of good performing markers for cervical cancer and seen that several combinations provided a sensitivity and also for HPV typing. specificity greater than 90%. TABLE 9 Summary of the results obtained for all the tested markers on scraping samples from cervical cancer patients and from control patients (Sens: sensitivity; SCC: Squamous cell carcinoma; Ade: adenocarcinoma; cncr: cancer; ctrl: control). NID2 CDO1 CDO1 LMX1A TAC1 GREM1 HOXA11 JAM3 9091 55928 55929 9513 561.87 29777 23844 JPH3

Sensitivity 81.0% 78.59% 82.3% 78.59% 75.9% 72.2% 72.2% 62.0% 64.6% Specificity 98.6% 98.6% 95.7% 97.1% 97.1% 98.6% 97.1% 100.0% 98.6% Sens SCC 80.3% 83.3% 81.8% 77.3% 77.3% 72.7% 72.7% 59.1% 69.7% Sens Ade 84.6% 53.8% 84.6% 84.6% 69.2% 69.2% 69.2% 76.9% 38.5% cncr test- 64 62 65 62 60 57 57 49 51 cncr test- 15 17 14 17 19 22 22 30 28 ctrl test- 1 1 3 2 2 1 2 O 1 ctrl test- 68 68 66 67 67 68 67 69 68 SCC test- 53 55 S4 51 51 48 48 39 46 SCC test- 13 11 12 15 15 18 18 27 2O Ade test- 11 7 11 11 9 9 9 10 5 Ade test- 2 6 2 2 4 4 4 3 8 cncri ctrl 4.75E-26 1.21E-24 4.57E-24 3.11E-23 6.24E-22, 186E-21 4.17E-2O 1.23E-18, 4.12E-18 p-val 5.32E-O1 2.31E-O2 5.84E-O1 4.33E-O1 3.37E-O1 4.79E-01 4.79E-O1 186E-O1 3.53E-O2 AdefSCC Cut off 2 5 5 35 15 15 10 1 5

C13orf18 CCNA1 TERT NDRG2 NOL4 LAMA1 GATA-4 Gron Gron 23702 56.603 1964.5 63431

Sensitivity 62.0% 53.2% 51.9% 58.2% 49.4% 43.0% 51.9% Specificity 97.1% 100.0% 100.0% 97.1% 98.6% 98.6% 94.2% Sens SCC 62.1% 54.5% 57.6% 60.6% 48.5% 43.9% SO.0% Sens Ade 61.5% 46.2% 23.1% 46.2% 53.8% 38.5% 61.5% cncr test- 49 42 41 46 39 34 41 cncr test- 30 37 38 33 40 45 38 ctrl test- 2 O O 2 1 1 4 ctrl test- 67 69 69 67 68 68 65 SCC test- 41 36 38 40 32 29 33 SCC test- 25 30 28 26 34 37 33 Ade test- 8 6 3 6 7 5 8 Ade test- 5 7 10 7 6 8 5 cncri ctrl 7.73E-16 2.91E-15 8.21E-15 2.03E-14 159E-12 1.61E-10 217E-10 p-val 6.43E-O1 4.OOE-O1 2.33E-O2 2.54E-O1 4.8OE-01 4.81E-01 3.25E-01 AdefSCC Cut off 2 O 1 5 150 5 10 US 9,371,569 B2 67 68 TABLE 10 Examples of the performance of combination of markers on scraping samples from cervical cancer patients and from control patients (Sens: sensitivity; SCC: Squamous cell carcinoma; Ade: adenocarcinoma; cncr: cancer: ctrl: control). JAM38, JAM38, JAM3% CDO1, 55929% JAM38, NID2 90918 TAC1561878. NID2 9091X HOXA11 23844% JAM3% HOXA11 23844% HOXA11 23844% HOXA11 23844% HOXA11 23844 CCNA1 Gron HOXA11 23844 GREM1. 29777 CDO1, 55929 CDO1, 55929

Sensitivity 89.9% 92.4% 88.6% 91.1% 92.4% 92.4% Specificity 98.6% 95.7% 98.6% 95.7% 94.2% 94.2% Sens SCC 92.4% 92.4% 89.4% 92.4% 92.4% 92.4% Sens Ade 76.9% 92.3% 84.6% 84.6% 92.3% 92.3% cncr test- 71 73 70 72 73 73 cncr test- 8 6 9 7 6 6 ctrl test- 1 3 1 3 4 4 ctrl test- 68 66 68 66 65 65 SCC test- 61 61 59 61 61 61 SCC test- 5 5 7 5 5 5 Ade test- 10 12 11 11 12 12 Ade test- 3 1 2 2 1 1 p-val 814E-32 6.6OE-31 6.87E-31 6.62E-30 1.17E-29 1.17E-29 cncri ctrl

JAM38, HOXA11 23844% JAM3% JAM38, NID2 90918 CDO1, 55929 CDO1, 55928 NID2 9091 CDO1, 55928 Sensitivity 92.4% 89.9% 86.1% 88.6% Specificity 94.2% 94.2% 97.1% 94.2% Sens SCC 92.4% 89.4% 86.4% 89.4% Sens Ade 92.3% 92.3% 84.6% 84.6% cncr test- 73 71 68 70 cncr test- 6 8 11 9 ctrl test- 4 4 2 4 ctrl test- 65 65 67 65 SCC test- 61 59 57 59 SCC test- 5 7 9 7 Ade test- 12 12 11 11 Ade test- 1 1 2 2 p-val 1.17E-29 9.85E-28 1.13E-27 7.67 E-27 cncri ctrl

HPV testing will certainly continue to occupy a significant indicating CIN 1 or CIN 0 after cytological examination. position in the diagnosis of cervical cancer. With this in mind, Overall the specificity of the methylation markers was higher the best performing methylation markers were tested on 40 compared to hr-HPV or HPV 16 testing but with a lower scraping samples from patients who were referred to the sensitivity. Combinations of methylation markers (where at hospital with an abnormal Pap Smear and these samples were least one of the markers scores positive) showed a comparable also tested for hr HPV and HPV16. The provisional cut off as sensitivity and specificity for cancers and controls, but a much defined above was reduced in order to obtain the highest higher specificity for CINO and CIN1. The sensitivity for possible sensitivity and specificity compared to the perfor 45 CIN3 and CIN2 is however somewhat lower. In order to mance ofhrHPV. The results of these tests are shown in Table increase the sensitivity for CIN3 and CIN2 detection, an 11. For these testing, the classification of pre-cancerous analysis was made of combining the results of methylation (CIN) conditions were used. Sensitivity was calculated for markers and HPV 16 (Table 12). The sensitivity as well as the samples indicating cancer, CIN 2 and CIN3, while specificity specificity increased if HPV 16 was combined with the was calculated for those samples from controls, and those methylation markers. TABLE 11 Overall Summary of the methylation marker(s) results on scraping samples from patients who were referred to the hospital with an abnormal Pap Smear, and from cervical cancer and control patients. (Sens: sensitivity; Spec: specificity: CINO, CIN1, CIN2, CIN3: cervical intraepithelial neoplasia grade 0, 1, 2, and 3; cncr: cancer, ctrl: control, NA: not applicable). hir-HPV HPV 16 JAM3 NID2 9091 LMX1A 9513 CDO1, 55928 TAC1561.87 C13ORF18 Gron

Sens Cncr 90% 779, 83% 80% 82% 83% 7396 S4% Sens CIN3 95% 83% 38% 40% 60% 43% 1796 24% Sens CIN2 74% 45% 21% 29% 29% 24% 79% 59% Spec Cntrl 96% 99% 99% 93% 94% 91% 93% 100% Spec CINO 51% 91% 98% 95% 91% 98% 100% 100% Spec CIN1 34% 78% 98% 93% 85% 88% 100% 98% Overall 87% 70% 56% 57% 63% S8% 42% 34% SelS Overall 67% 91% 98% 93% 91% 92% 97% 99% spec Cut off NA NA 1 2 10 3 10 O US 9,371,569 B2 69 70 TABLE 1 1-continued Overall Summary of the methylation marker(s) results on scraping samples from patients who were referred to the hospital with an abnormal Pap Smear, and from cervical cancer and control patients. (Sens: sensitivity; Spec: specificity; CINO, CIN1, CIN2, CIN3: cervical intraepithelial neoplasia grade 0, 1, 2, and 3; cncr: cancer: ctrl: control, NA: not applicable). cncr test- 74 63 68 66 67 68 60 44 cncr test- 8 19 14 16 15 14 22 38 CIN3 test- 40 35 16 17 25 18 7 10 CIN3 test- 2 7 26 25 17 24 35 32 CIN2 test- 31 19 9 12 12 10 3 2 CIN2 test- 11 23 33 30 30 32 39 40 ctrl test- 3 1 1 5 4 6 5 O ctrl test- 66 68 68 64 65 63 64 69 CINO test- 21 4 1 2 4 1 O O CINO test- 22 39 42 41 39 42 43 43 CIN1 test- 27 9 1 3 6 5 O 1 CIN1 test- 14 32 40 38 35 36 41 40

JAM3% JAM38, JAM38, JAM38, CDO1, 55928% NID2X NID2 9091 LMX1A 9513 CDO1, 55928 NID2 9091 LMX1A 9513

Sens Cncr 88% 87% 90% 91% 88% Sens CIN3 45% 62% 52% 57% 67% Sens CIN2 33% 36% 31% 38% 43% Spec Cntrl 93% 93% 90% 84% 88% Spec CINO 95% 93% 98% 95% 91% Spec CIN1 93% 83% 88% 83% 83% Overall 63% 67% 66% 69% 71.9% SelS Overall 93% 90% 92% 87% 88% spec Cuto NA NA NA NA NA cncr test- 72 71 74 75 72 cncr test- 10 11 8 7 10 CIN3 test- 19 26 22 24 28 CIN3 test- 23 16 2O 18 14 CIN2 test 14 15 13 16 18 CIN2 test- 28 27 29 26 24 ctrl test- 5 5 7 11 8 ctrl test- 64 64 62 58 61 CINO test- 2 3 1 2 4 CINO test- 41 40 42 41 39 CIN1 test- 3 7 5 7 7 CIN1 test- 38 34 36 34 34

TABLE 12 Overall summary results of methylation marker(s) in combination with HPV 16 on scraping samples from patients who were referred to the hospital with an abnormal Pap Smear, and from cervical cancer and control patients. (Sens: sensitivity; Spec: specificity: CINO, CIN1, CIN2, CIN3: cervical intraepithelial neoplasia grade 0, 1, 2, and 3; cncr: cancer: ctrl: control). JAM38 NID2 9091X LMX1A 95138. CDO1, 559288 PAC1561878 C13 ORF18 Gronx hir-HPV HPV 16 HPV16 HPV16 HPV16 HPV16 HPV 16 HPV16

Sens Cncr 90% 770, 95% 93% 94% 99% 93% 83% Sens CIN3 95% 83% 88% 90% 88% 88% 83% 86% Sens CIN2 74% 45% 60% 62% 60% 60% SO% 45% Spec Cntrl 96% 99% 97% 93% 93% 90% 91% 99% Spec CINO 51% 91% 88% 86% 84% 88% 91% 91% Spec CIN1 34% 78% 78% 7396 66% 7396 78% 76% CINO test- 22 39 38 37 36 38 39 39 CINO test- 21 4 5 6 7 5 4 4 CIN1 test- 14 32 32 30 27 30 32 31 CIN1 test- 27 9 9 11 14 11 9 10 CIN2 test- 11 23 17 16 17 17 21 23 CIN2 test- 31 19 25 26 25 25 21 19 CIN3 test- 2 7 5 4 5 5 7 6 CIN3 test- 40 35 37 38 37 37 35 36 cncr test- 8 19 4 6 5 1 6 14 cncr test- 74 63 78 76 77 81 76 68 ctrl test- 66 68 67 64 64 62 63 68 ctrl test- 3 1 2 5 5 7 6 1 Overall 87% 70% 84% 84% 84% 86% 80% 74% SelS Overall 67% 91% 90% 86% 83% 85% 88% 90% spec US 9,371,569 B2 71 72 TABLE 12-continued Overall summary results of methylation marker(s) in combination with HPV 16 on scraping samples from patients who were referred to the hospital with an abnormal Pap Smear, and from cervical cancer and control patients. (Sens: sensitivity; Spec: specificity: CINO, CIN1, CIN2, CIN3: cervical intraepithelial neoplasia grade 0, 1, 2, and 3; cncr: cancer: ctrl: control). JAM3% JAM38, JAM3% JAM38, CDO1, 55928% NID2X NID2 9091X LMX1A 95138. CDO1, 55928% NID2 9091X LMX1A 95138. HPV16 HPV16 HPV16 HPV 16 HPV16

Sens Cncr 98% 96% 100% 100% 96% Sens CIN3 90% 88% 88% 90% 90% Sens CIN2 67% 64% 64% 67% 67% Spec Cntrl 93% 91% 88% 84% 88% Spec CINO 86% 84% 88% 86% 81% Spec CIN1 7396 66% 7396 68% 66% CINO test- 37 36 38 37 35 CINO test- 6 7 5 6 8 CIN1 test- 30 27 30 28 27 CIN1 test- 11 14 11 13 14 CIN2 test- 14 15 15 14 14 CIN2 test- 28 27 27 28 28 CIN3 test- 4 5 5 4 4 CIN3 test- 38 37 37 38 38 cncr test- 2 3 O O 3 cncr test- 8O 79 82 82 79 ctrl test- 64 63 61 58 61 ctrl test- 5 6 8 11 8 Overall 88% 86% 88% 89% 87% SelS Overall 86% 82% 84% 80% 80% spec

As cytology is currently been used and hr-HPV testing has 30 using HPV testing in primary Screening has been tempered by been Suggested as primary Screening tool in population-based its somewhat poorer PPV (19%) in comparison with cytologi cervical screening, we simulated the effect on the perfor cal analysis (27%). Using methylation as triage test, the PPVs mances of the methylation tests if only cytology (Table 13) or were much higher. hr-HPV (Table 14) positive patients were selected. The triage Taking the limitations of cytology and the decreased dis 35 ease prevalence due to the introduction of HPV vaccination simulations were based on the performance results obtained programs into account, it is proposed to use a highly sensitive in Table 11 and Table 12. The performance of cytology and and objective screening test such as HPV DNA testing to hr-HPV testing were based on data from literature. identify the rare cases of cancer precursors and to combine it, The performances of the triage tests showed much higher when positive, with another test which has a high degree of specificity resulting infewer referrals for colposcopy than did specificity, such as methylation testing. Moreover, methyla cytology or hr-HPV testing alone but were less sensitive. 40 tion is measuring changes in the host cells, as precursor of Testing for hr-HPV types has a higher sensitivity for detecting cervix cancer, while HPV is detecting the causative agent. CIN2+ than cytology. The NPV is close to 100% thus allow This is an ideal methodology for a screening and a triage ing for less frequent screening and longer screening intervals assay because they should measure different but complemen without jeopardizing patients’ safety. But, the enthusiasm for tary biological signals. TABLE 13 The simulation of the performance of Cytology test as a first-line screening test on 70000 women and the methylation marker test(s) in-or excluding HPV 16 as triage test. (Sens: sensitivity: Spec: specificity; CINO/1, CIN2+: cervical intraepithelial neoplasia grade O and 1, and grade

2 and 3 and cancers: PPV: positive predictive value: NPV: negative predictive value). Cytology, Cytology, Cytology, Cytology, Triage Cytology, Cytology, Triage Triage Triage NID2X Triage hr- Triage JAM38, JAM38, NID2X LMX1AX Cytology HPV HPV16 NID2 NID2WHPV 16 LMX1A HPV16

CIN2+ Test S4O 449 334 213 417 290 417 CIN2-. Test- 230 321 436 557 353 480 353 CINO.1 Test- 1460 825 219 85 288 187 374 CINO.1 Test- 67770 684OS 69.011 69145 68.942 69043 68856 Sens 70.1% 58.3% 43.4% 27.6% 54.2% 37.7% 54.2% Spec 97.9% 98.8% 99.7% 99.9% 99.6% 99.7% 99.5% NPV 99.7% 99.9% 99.7% 99.5% 99.8% 99.6% 99.8% PPV 27.0% 35.2% 60.4% 71.4% 59.1% 60.8% 52.7% Colposcopy 2OOO 1274 553 298 706 477 791 referrals US 9,371,569 B2 73 74 TABLE 1.4 The simulation of the performance of hr-HPV test as a first-line screening test on 70000 women and the methylation marker test(s) in-or excluding HPV 16 as triage test. (Sens: sensitivity: Spec: specificity: CINO/1, CIN2+: cervical intraepithelial neoplasia grade O and 1, and ade 2 and 3 and cancers: PPV: positive predictive value: NPV: negative predictive value).

hr-HPV, hr-HPV, hr-HPV, Triage hr-HPV, Triage hr-HPV, hr-HPV, Triage JAM3% Triage NID2X Triage Triage JAM38, NID2X NID2X LMX1AX hr-HPV Cytology HPV16 NID2 HPV16 LMX1A HPV16 CIN2+ Test- 665 532 433 276 541 376 541 CIN2-. Test- 67 285 333 491 226 390 226 CINO.1 Test- 2835 553 420 164 553 358 717 CINO.1 Test- 66433 68631 68814 69070 68681 68875 68517 Sens 90.8% 65.1% 56.5% 36.0% 70.6% 49.1% 70.6% Spec 95.9% 99.2% 99.4% 99.8% 99.2% 99.5% 99.0% NPV 99.9% 99.6% 99.6% 99.4% 99.8% 99.5% 99.8% PPV 19.0% 49.0% SO.8% 62.7% 49.5% 51.2% 43.0% Colposcopy 3500 1085 853 440 1094 735 1258 referrals

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Papillomavirus Type 16 DNA in Asymptomatic Infection 65 Thomassin H. et al. MethylOuant: a sensitive method for and Cervical Neoplasia, Journal of Virology, December quantifying methylation of specific cytosines within the 2004, p. 12762-12772. genome. 2004. Nuc Acid Res 32, 21. US 9,371,569 B2 75 76 Thompson J D, Higgins D G, Gibson T. J. CLUSTAL W: van Dongen, J. J. et al. (2003) Design and standardization of improving the sensitivity of progressive multiple sequence PCR primers and protocols for detection of clonal immu alignment through sequence weighting, position-specific noglobulin and T-cell receptor gene recombinations in Sus gap penalties and weight matrix choice. Nucleic Acids Res pect lymphoproliferations: report of the BIOMED-2 Con 1994; 22(22):4673-80. certed Action BMH4-CT98-3936. Leukemia, 17, 2257 2317. Tost, J. et al. Analysis and accurate quantification of CpG Virmani et al. (2001) Aberrant methylation during cervical methylation by MALDI mass spectrometry. Nuc. Acid carcinogenesis. Clin Cancer Research, March; 7(3):584-9. Res, 2003, 31(9): e50. Xiong, Z. G. and Laird, P. W. (1997) COBRA: A sensitive and Trinh B. et al. DNA methylation analysis by Methylight quantitative DNA methylation assay. Nucleic Acids technology. Methods. 2001 December; 25(4). Research, 25, 2532-2534. Trooskens G. De Beule D, Decouttere F, Van Criekinge W. Zeschnigk M. et al. A novel real-time PCR assay for quanti Phylogenetic trees: visualizing, customizing and detecting tative analysis of methylated alleles (QAMA): analysis of incongruence. Bioinformatics 2005; 21 (19):3801-2. the retinoblastoma locus. 2004. Nuc Acid Res 32, 16.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 449

<21 Os SEQ ID NO 1 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs SEQUENCE: 1

gtttggttcg ggittagcgt. 19

<21 Os SEQ ID NO 2 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs SEQUENCE: 2

ttgcgttitta tttgt atttic go 22

<21 Os SEQ ID NO 3 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs SEQUENCE: 3

ttt tattgcg agtcgt.cggit c 21

<21 Os SEQ ID NO 4 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs SEQUENCE: 4

tgtataggag togaagggac gta 23

<21 Os SEQ ID NO 5 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs SEQUENCE: 5

gttaggtaag togtacggcg a 21 US 9,371,569 B2 77 78 - Continued

<210s, SEQ ID NO 6 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 6 atttaatgag gacgg taggit agc 23

<210s, SEQ ID NO 7 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 7 tittaatataa gtcgggittac gttcg 25

<210s, SEQ ID NO 8 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 8 ttagggagta agtgcgtttg C 21

<210s, SEQ ID NO 9 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 9 tagacggitta cagtaggcg gta 23

<210s, SEQ ID NO 10 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 10 tttitt.cgitat tittaggaagt gigc 23

<210s, SEQ ID NO 11 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 11 tittggggttc gattatattt c 21

<210s, SEQ ID NO 12 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: US 9,371,569 B2 79 80 - Continued <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 12 gttatttitt C ggcgggttc 19

<210s, SEQ ID NO 13 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 13 tacgcgtagg ttittaagt cq c 21

<210s, SEQ ID NO 14 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 14 tittgatttitt gaaag.cgt.cg t 21

<210s, SEQ ID NO 15 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 15 tttittaggga agtaaag.cgt. c9 22

<210s, SEQ ID NO 16 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 16 tagtttggta gttagcgggit c 21

<210s, SEQ ID NO 17 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 17 at agggggag titcggtacg 19

<210s, SEQ ID NO 18 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 18 cgtttittata gggittittggit tac 25 US 9,371,569 B2 81 82 - Continued

<210s, SEQ ID NO 19 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 19 tagttgtatic ggtttaggcg titt 23

<210s, SEQ ID NO 2 O &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2O gttatggcga tigcggttt C 19

<210s, SEQ ID NO 21 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 21 gaaggtagcg tttitt.cgatg

<210 SEQ ID NO 22 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 22 aattittaggit tagagggitta t cqc 24

<210s, SEQ ID NO 23 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 23 gagggggtag gaaagttcgc 19

<210s, SEQ ID NO 24 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 24 gggacgattt titcgttgttc

<210s, SEQ ID NO 25 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 9,371,569 B2 83 84 - Continued

<4 OOs, SEQUENCE: 25 aattitcgttt gtagagtc.gt cqt 23

<210s, SEQ ID NO 26 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 26 gtcggtaagg tttggaga.gc

<210s, SEQ ID NO 27 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 27 ttagaagtaa tittaggcgcg titc 23

<210s, SEQ ID NO 28 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer < 4 OO SEQUENCE: 28 aatttgattt gtgttgttgtat cqc 23

<210s, SEQ ID NO 29 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 29 gtttacgcga tttittgggac

<210s, SEQ ID NO 3 O &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 30 agggitttitt C ggagtcgtt 19

<210s, SEQ ID NO 31 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 31 aggcgt.cgta tittatagcgt tt 22

<210s, SEQ ID NO 32 &211s LENGTH: 2O US 9,371,569 B2 85 86 - Continued

&212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 32 gtgggggtc.g gtgtagtatic

<210s, SEQ ID NO 33 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 33 tataaaaggg gttcgggitta gtc 23

<210s, SEQ ID NO 34 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 34 aggaagtatt tattgcgitat gtttc 25

<210s, SEQ ID NO 35 &211s LENGTH: 23 & 212 TYPE DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 35 taattittaag gaggacgagg gtc 23

<210s, SEQ ID NO 36 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 36 gttittgggag aggcggttc 19

<210s, SEQ ID NO 37 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 37 ggtgtagcgt ttagggtcgt C 21

<210s, SEQ ID NO 38 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 38 US 9,371,569 B2 87 88 - Continued ggatagt cq9 atcgagittaa C9tc 24

<210s, SEQ ID NO 39 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 39 aggataggta taattitcgg ttt C 24

<210s, SEQ ID NO 4 O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 4 O cgggtttgag ggtaatagaa ticg 23

<210s, SEQ ID NO 41 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 41 gtgcggggta agaaggaac 19

<210s, SEQ ID NO 42 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 42 gaga.gaga aa gC9ggagttc

<210s, SEQ ID NO 43 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 43 taggagcgtt gtttcggit c 19

<210s, SEQ ID NO 44 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 44 tittaggtggg aag.cgt attt atc 23

<210s, SEQ ID NO 45 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial US 9,371,569 B2 89 90 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 45 gggatagtgg gttgacgc 19

<210s, SEQ ID NO 46 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 46 gcgtgggttt t cqtcgtag 19

<210s, SEQ ID NO 47 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 47 cgggttgtag ttaat atcga gg 22

<210s, SEQ ID NO 48 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial & 22 O FEATURE; <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 48 tttgttcgtt titt cqattgt to 22

<210s, SEQ ID NO 49 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 49 cgittaatcgg atalaga.gtgc g 21

<210s, SEQ ID NO 50 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 50 gtatagitttc gtagtttgcg tittagc 26

<210s, SEQ ID NO 51 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 51 gg tattgtta ttittgcgttt to 22 US 9,371,569 B2 91 92 - Continued

<210s, SEQ ID NO 52 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 52 gattitcgggt tt tatggc 19

<210s, SEQ ID NO 53 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 53 gaaagaagga ggtttcggc 19

<210s, SEQ ID NO 54 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 54 gttgtgagtt gcgttttitta cqtc 24

<210s, SEQ ID NO 55 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 55 ggtcgtag to gtag toggg 19

<210s, SEQ ID NO 56 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 56 gaatttggta cattttacg gag 23

<210s, SEQ ID NO 57 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 57 atttag tatt gggggggagc

<210s, SEQ ID NO 58 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 9,371,569 B2 93 94 - Continued

<4 OOs, SEQUENCE: 58 agtag tagga atagaaacgg Ca 23

<210s, SEQ ID NO 59 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 59 gaagttggitt agggitacggit c 21

<210s, SEQ ID NO 60 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 60 ggat.cgttgg attittggttc

<210s, SEQ ID NO 61 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 61 tttittagagt aaatagcggg agc 23

<210s, SEQ ID NO 62 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 62 ttittattt at t cqgggagtt gc 22

<210s, SEQ ID NO 63 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 63 tcgtagggitt C9tagt cqtt t 21

<210s, SEQ ID NO 64 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 64 gtcggttgac gttittgagat aagtic 25

<210s, SEQ ID NO 65 US 9,371,569 B2 95 96 - Continued

&211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 65 tcqgattt cq tttittagcgt at 22

<210s, SEQ ID NO 66 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 66 taggttggitt tdttt C9gt C 21

<210s, SEQ ID NO 67 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 67 gggattataa gtc.gcgtc.gc

<210s, SEQ ID NO 68 <211 LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 68 ttagattt cq taaacggtga aaac 24

<210s, SEQ ID NO 69 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 69 tggatggagt ttaggittata t catc 25

<210s, SEQ ID NO 70 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 7 O aggagggatt gtcggattta C 21

<210s, SEQ ID NO 71 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 71 US 9,371,569 B2 97 98 - Continued tttittagatt tat cagtgg C9 22

<210s, SEQ ID NO 72 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 72 cggitat cqtt gtttaggagg C 21

<210s, SEQ ID NO 73 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 73 agttgtttgg tattogcggit

<210s, SEQ ID NO 74 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 74 gttagattga titt cqttcga gg 22

<210s, SEQ ID NO 75 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 75 tagtagattt ttagcggtga agacg 25

<210s, SEQ ID NO 76 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 76 titcgggttitt tttgtttitta attic 24

<210s, SEQ ID NO 77 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 77 gtaagtgagt titcgagtgtc gc 22

<210s, SEQ ID NO 78 &211s LENGTH: 21 &212s. TYPE: DNA US 9,371,569 B2 99 100 - Continued

<213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 78 gaaaggtogg atttgtttitt C 21

<210s, SEQ ID NO 79 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 79 agattttgttg gtttcqtcgt t 21

<210s, SEQ ID NO 8O &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 80 gcqgtttitta aggagttitta tttitc 25

<210s, SEQ ID NO 81 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 81 gagagatt.cg ggatt.cgtg 19

<210s, SEQ ID NO 82 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 82 agtacgttgt titcggagttt titc 23

<210s, SEQ ID NO 83 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 83 gcgt.cgttitt gtatgggt at C 21

<210s, SEQ ID NO 84 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 84 cgagtagtag ttgcgt.cggg US 9,371,569 B2 101 102 - Continued

<210s, SEQ ID NO 85 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 85 tgtatgattt tagttcgcgg at 22

<210s, SEQ ID NO 86 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 86 gcgggattta tttgttacgg a 21

<210s, SEQ ID NO 87 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 87 attgcgt.cgg gtttagtttc

<210s, SEQ ID NO 88 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 88 gtttagggaa agcggacga 19

<210s, SEQ ID NO 89 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 89

19

<210s, SEQ ID NO 90 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 90 ttattittgcg agcggttt c 19

<210s, SEQ ID NO 91 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: US 9,371,569 B2 103 104 - Continued <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 91 gatttgggcg tttittggittt titcgc 25

<210s, SEQ ID NO 92 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 92 gtgggittitta agtttacggit titc 23

<210s, SEQ ID NO 93 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 93 ggcggagttt gtatagaggc

<210s, SEQ ID NO 94 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 94 tttittgggitt togttgtttc

<210s, SEQ ID NO 95 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 95 titcgtttcgt ttaggitat cq titt 23

<210s, SEQ ID NO 96 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 96 tittaggatta tagtgagcga C9g 23

<210s, SEQ ID NO 97 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 97 gcgttgaagt cqgggttc 18 US 9,371,569 B2 105 106 - Continued

<210s, SEQ ID NO 98 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 98 ggtcgttt cq ttgttittata gc 22

<210s, SEQ ID NO 99 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 99 ttacggittag tagaaggagt agcgt. 25

<210s, SEQ ID NO 100 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 1.OO tcgaggaaga agatgtcgaa g 21

<210 SEQ ID NO 101 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 101 gaggcgtaag taggcgaaa 19

<210s, SEQ ID NO 102 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 102 gattagagcg agcgaacga 19

<210s, SEQ ID NO 103 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 103 ggtttgttgg togtttittag C 21

<210s, SEQ ID NO 104 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 9,371,569 B2 107 108 - Continued

<4 OOs, SEQUENCE: 104 gtatttaggg tagcgggtc.

<210s, SEQ ID NO 105 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 105 gcgittatggt gtttittatag ct 23

<210s, SEQ ID NO 106 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 106 ttgtaggcgg tttgtagt cq t 21

<210s, SEQ ID NO 107 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer < 4 OO SEQUENCE: 107 gaggat.cggt ttaggttgc 19

<210s, SEQ ID NO 108 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 108 aggggalagac galaga.gc.gt 19

<210s, SEQ ID NO 109 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 109 tagcggagag gagattacgc

<210s, SEQ ID NO 110 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 110 agggg tattt at aggcgttt agc 23

<210s, SEQ ID NO 111 &211s LENGTH: 21 US 9,371,569 B2 109 110 - Continued

&212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 111 gaagggitaat cqggtgttitt C 21

<210s, SEQ ID NO 112 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 112 gatagggittt tttitt.cgc

<210s, SEQ ID NO 113 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 113 ttgtagttitt cagttggag gtc 23

<210s, SEQ ID NO 114 &211s LENGTH: 21 & 212 TYPE DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 114 gttaggagtt C9tcggittag C 21

<210s, SEQ ID NO 115 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 115 gagatgtttic gagggttgc 19

<210s, SEQ ID NO 116 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 116 titt.cgcggitt ttittagattg titc 23

<210s, SEQ ID NO 117 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 117 US 9,371,569 B2 111 112 - Continued atcgtaggitt gggitttggtc

<210s, SEQ ID NO 118 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 118 tggttgcgtt gttitat cott t 21

<210s, SEQ ID NO 119 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 119 ggg tattt at ticgacggat

<210s, SEQ ID NO 120 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 120 ggttt Cata gC9tagttgt titc 23

<210s, SEQ ID NO 121 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 121 gttctgttggg taaggcgttc

<210s, SEQ ID NO 122 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 122 taagga attt ttatt.cgga ggc 23

<210s, SEQ ID NO 123 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 123 gtggittagcg gattitcgagt

<210s, SEQ ID NO 124 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial US 9,371,569 B2 113 114 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 124 titt.cgtaggg ttcggtgtc. 19

<210s, SEQ ID NO 125 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 125 gttagggttc ggggg.cgttg tt 22

<210s, SEQ ID NO 126 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 126 tittagttcgt tagttt cqtc ggit 23

<210s, SEQ ID NO 127 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial & 22 O FEATURE; <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 127 ttttgtggitt agt cqcggit 19

<210s, SEQ ID NO 128 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 128 gcqt cqttag at attttgtt gc 22

<210s, SEQ ID NO 129 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 129 gtatggagcg ttittgcgat 19

<210s, SEQ ID NO 130 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 130 tgtgttatat cqgittagttg agagc 25 US 9,371,569 B2 115 116 - Continued

SEQ ID NO 131 LENGTH: 22 TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 131 cgtttgttitt tataggttcg gg 22

SEQ ID NO 132 LENGTH: 2O TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 132 tgtatgcgga gaggtogtag

SEQ ID NO 133 LENGTH: 21 TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 133 cc tactitatic tict cocqctic g 21

SEQ ID NO 134 LENGTH: 23 TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 134 cittaacgaac gacitta accq act 23

SEO ID NO 135 LENGTH: 22 TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 135 tataccgaac titat cqcctic cq 22

SEQ ID NO 136 LENGTH 19 TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer

SEQUENCE: 136 aaacaact co gaacgacga 19

SEO ID NO 137 LENGTH: 23 TYPE: DNA ORGANISM: artificial FEATURE: OTHER INFORMATION: primer US 9,371,569 B2 117 118 - Continued

<4 OOs, SEQUENCE: 137 aaaaacgact acaactacga cqa 23

<210s, SEQ ID NO 138 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 138 aacaaact cq cittctacacg aa 22

<210s, SEQ ID NO 139 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 139 atacgacgca aaaactat cq c 21

<210s, SEQ ID NO 140 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 140 aaaaac cqat taacctacgc tic 22

<210s, SEQ ID NO 141 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 141 ccttcttaaa acgacgacga a 21

<210s, SEQ ID NO 142 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 142 titcc toccga acctttacga

<210s, SEQ ID NO 143 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 143 cgaaaactico gaalaccgat 19

<210s, SEQ ID NO 144 US 9,371,569 B2 119 120 - Continued

&211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 144 ctaataatcg ccc ctitcgc 19

<210s, SEQ ID NO 145 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 145 tcc.cgaacta aacgaaac cc cq 22

<210s, SEQ ID NO 146 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 146 acaccacgca cctatacgc 19

<210s, SEQ ID NO 147 <211 LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 147 acgtaatact aaaccogaac go 22

<210s, SEQ ID NO 148 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 148 aaacct coga aataac cqtc

<210s, SEQ ID NO 149 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 149 acaatttacc cc.gct cqact

<210s, SEQ ID NO 150 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 150 US 9,371,569 B2 121 122 - Continued aaatct cqaa act caccitaa cqa 23

<210s, SEQ ID NO 151 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 151 ctacct tcgt accctt cqat t 21

<210s, SEQ ID NO 152 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 152

CCalacctaaa. aaacgaccga

<210s, SEQ ID NO 153 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 153 aaataaac cc gatcc.gcaa. 19

<210s, SEQ ID NO 154 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 154 accalatcaac aacg.cgaac 19

<210s, SEQ ID NO 155 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 155 cgaaacgacc taaaaacctic g 21

<210s, SEQ ID NO 156 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 156 ttctac tact citcgct ct co gac 23

<210s, SEQ ID NO 157 &211s LENGTH: 23 &212s. TYPE: DNA US 9,371,569 B2 123 124 - Continued

<213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 157 t citat attaa aaactt cqct tcg 23

<210s, SEQ ID NO 158 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 158 aaaataacaa aaccc.gtc.cg

<210s, SEQ ID NO 159 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 159 aaac ccc.gta caataacca

<210s, SEQ ID NO 160 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 160 gaaacgtaaa aatat cqt cq ca 22

<210s, SEQ ID NO 161 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 161 aaaaac ccta cqaacacgac t 21

<210s, SEQ ID NO 162 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 162 titcc to aacc gtc.t.ccacg 19

<210s, SEQ ID NO 163 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 163 tcc.cct tact titcc.gcgac 19 US 9,371,569 B2 125 126 - Continued

<210s, SEQ ID NO 164 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 164 t ccc tactaa aaacgc.cgaa

<210s, SEQ ID NO 165 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 165 aaattaacgt. c cqct catac g 21

<210s, SEQ ID NO 166 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 166 c cat cactta t cotcgacgc

<210s, SEQ ID NO 167 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 167 ataaacticca acgacgcgaa a 21

<210s, SEQ ID NO 168 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 168 t cataataac gaaacgacga cc 22

<210s, SEQ ID NO 169 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 169 cgaatttitt c ctacgtaacc g 21

<210s, SEQ ID NO 170 &211s LENGTH: 16 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: US 9,371,569 B2 127 128 - Continued <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 170 c cct cocaaa cqc cqa 16

<210s, SEQ ID NO 171 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 171 aaacgaacga acaacaacga

<210s, SEQ ID NO 172 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 172 cgc.cacaata acgt.cgaaa 19

<210s, SEQ ID NO 173 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 173 aaaaacaatc aaatacgaaa cqc 23

<210s, SEQ ID NO 174 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 174 t cacaattac ccc.gaaacg 19

<210s, SEQ ID NO 175 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 175 cacgaccc cc taactic cqt 19

<210s, SEQ ID NO 176 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 176 aaacgctaaa accogcaat 19 US 9,371,569 B2 129 130 - Continued

<210s, SEQ ID NO 177 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 177 ataaaaatcc cqacgaacga

<210s, SEQ ID NO 178 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 178 cc caaaacta citcgcc.gct 19

<210s, SEQ ID NO 179 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 179 citct ct catt citacgc.cgitt c 21

<210 SEQ ID NO 18O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 18O taacgctata aaactic ctac cqc 23

<210s, SEQ ID NO 181 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 181 aaaaccacgc gaaaaacga 19

<210s, SEQ ID NO 182 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 182 aact cqcgac togaatcc cc g 21

<210s, SEQ ID NO 183 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 9,371,569 B2 131 132 - Continued

<4 OOs, SEQUENCE: 183 cc.cgaaacaa act acacgac

<210s, SEQ ID NO 184 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 184 ctaacttaac cqcatcgctic

<210s, SEQ ID NO 185 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 185 cc.cgataaat aataac attc acga 24

<210s, SEQ ID NO 186 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer < 4 OO SEQUENCE: 186 cgct accqat atcc.gctaaa cq 22

<210s, SEQ ID NO 187 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 187 cc.gcaaaaac ctaaaacgta a 21

<210s, SEQ ID NO 188 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 188 atctaaactt toccitat cqa cog 23

<210s, SEQ ID NO 189 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 189 taacgaaaac tacgacgacg a 21

<210s, SEQ ID NO 190 &211s LENGTH: 19 US 9,371,569 B2 133 134 - Continued

&212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 190 aaaact acaa cc.gc.cgaca 19

<210s, SEQ ID NO 191 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 191 aactitctitat acccgatcct cq 22

<210s, SEQ ID NO 192 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 192 tatat cotcg ccc.cacgtaa

<210s, SEQ ID NO 193 &211s LENGTH: 21 & 212 TYPE DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 193 atacgc.ctta caa.ccc ctac g 21

<210s, SEQ ID NO 194 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 194 acaaaatcct cqttct cqaa t 21

<210s, SEQ ID NO 195 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 195 tccaaatctt titt cogcoga 19

<210s, SEQ ID NO 196 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 196 US 9,371,569 B2 135 136 - Continued accotc.ttct cqaacgacg 19

<210s, SEQ ID NO 197 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 197 gaac acctac citcaaactaa cqac 24

<210s, SEQ ID NO 198 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 198 taaacgacga cct coat cq 19

<210s, SEQ ID NO 199 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 199 Cgaacgcaaa accgaaatcg

<210s, SEQ ID NO 2 OO &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2OO tctic ct coga aaaacgctic 19

<210s, SEQ ID NO 2 O1 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2O1 aaaatactac gaalacc.gc.cc

<210s, SEQ ID NO 202 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 2O2 gctic cqaatc aaaattaacg a 21

<210s, SEQ ID NO 203 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial US 9,371,569 B2 137 138 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2O3 cgaact cacc tict ctaccga c 21

<210s, SEQ ID NO 204 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 204 cgtataacta ttacct cqaa acgct 25

<210s, SEQ ID NO 205 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 205 cgaccc ct co taactitt.cg 19

<210s, SEQ ID NO 206 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial & 22 O FEATURE; <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2O6 aactaaaata ccc.gtact co got 23

<210s, SEQ ID NO 2 O7 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2O7 aaaaccittaa cqaaactaaa cqaaa 25

<210s, SEQ ID NO 208 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 208 gaaaac cata acgacgtact aacg 24

<210s, SEQ ID NO 209 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 209 citcc.gaaaac catacgc.cc 19 US 9,371,569 B2 139 140 - Continued

<210s, SEQ ID NO 210 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 210 accatctgat cacgcct cq 19

<210s, SEQ ID NO 211 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 211 atc.ccc.cgaa cattacgatt

<210s, SEQ ID NO 212 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 212 citacgaaatt coctittacgc t 21

<210s, SEQ ID NO 213 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 213 gtaatccalaa aataaaaact acgc.c 25

<210s, SEQ ID NO 214 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 214 citt catctac acct catac cc.g 23

<210s, SEQ ID NO 215 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 215 cc.cgataacc gctitcgitat 19

<210s, SEQ ID NO 216 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 9,371,569 B2 141 142 - Continued

<4 OOs, SEQUENCE: 216 acaaacgacc ctaaaaacga ac 22

<210s, SEQ ID NO 217 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 217 acgaattitta cct caaacga cc 22

<210s, SEQ ID NO 218 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 218 aaccc.gaaac tacgacitacg ac 22

<210s, SEQ ID NO 219 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 219 gcc cct tacc cataacgaac

<210s, SEQ ID NO 220 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 220 gaacgacaaa caaaact cqa aa 22

<210s, SEQ ID NO 221 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 221 gcqatccitat caaatc.cgaa

<210s, SEQ ID NO 222 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 222 gaatacticta attccacgcg act 23

<210s, SEQ ID NO 223 US 9,371,569 B2 143 144 - Continued

&211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 223 gactitct cat accogcaatcq

<210s, SEQ ID NO 224 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 224 cctaccct cq aaacaaacga

<210s, SEQ ID NO 225 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 225 aaccc.gaatt acgcaaacg 19

<210s, SEQ ID NO 226 & 211 LENGTH 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 226 cgcct caata ataccacc 19

<210s, SEQ ID NO 227 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 227 cct citcgatt coctacgttt

<210s, SEQ ID NO 228 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 228 taac cqcctt taaccocq a 19

<210s, SEQ ID NO 229 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 229 US 9,371,569 B2 145 146 - Continued cc.cgtact tc gctaactitta aacg 24

<210s, SEQ ID NO 230 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 23 O gcqttataca aataccc.ccg

<210s, SEQ ID NO 231 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 231 citacgaccaa actaaatc.cg aac 23

<210s, SEQ ID NO 232 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 232 aaaaac ccga acgaacgtaa

<210s, SEQ ID NO 233 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 233 cgcatctaca aacticcgaaa

<210s, SEQ ID NO 234 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 234 taac tactaa accc.gaac cq aac 23

<210s, SEQ ID NO 235 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 235 cgaalacat cq acacct tcgt.

<210s, SEQ ID NO 236 &211s LENGTH: 23 &212s. TYPE: DNA US 9,371,569 B2 147 148 - Continued

<213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 236 cgaaataaaa actaacaatc gcc 23

<210s, SEQ ID NO 237 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 237 tott cqataa citctaccc.cg a 21

<210s, SEQ ID NO 238 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 238 gacaat catc catcaatcga aa 22

<210s, SEQ ID NO 239 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 239 caattictaaa aacgcacgac t 21

<210s, SEQ ID NO 24 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 240 cacgaactaa cqctacgcaa.

<210s, SEQ ID NO 241 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 241 gacc cctaca tottaacaac cq 22

<210s, SEQ ID NO 242 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 242 tacct actico got accaacg taa 23 US 9,371,569 B2 149 150 - Continued

<210s, SEQ ID NO 243 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 243 ctaatt cqtc tat cocqtcc

<210s, SEQ ID NO 244 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 244 attittacc cc gctacct cq 19

<210s, SEQ ID NO 245 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 245 aaaacgatac gctaaaccc.g

<210s, SEQ ID NO 246 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 246 caccc.gaatt acaaat accq a 21

<210s, SEQ ID NO 247 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 247 cc.gcaat atc actaaacca

<210s, SEQ ID NO 248 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 248 catacct caa taacaaacaa acaaacg 27

<210s, SEQ ID NO 249 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: US 9,371,569 B2 151 152 - Continued <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 249 aaaaacaaaa cacgcgaaa 19

<210s, SEQ ID NO 250 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 250 ttacct actt coccg.cgac 19

<210s, SEQ ID NO 251 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 251 ccgacgacaa citaccgaaa 19

<210s, SEQ ID NO 252 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 252 ctacac ccta aaaacgcgaa c 21

<210s, SEQ ID NO 253 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 253 cataaaacga acaccc.gaac cq 22

<210s, SEQ ID NO 254 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 254 acctaacaaa citacgaacgc ca 22

<210s, SEQ ID NO 255 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 255 aactaaacaa cacticcgaac ga 22 US 9,371,569 B2 153 154 - Continued

<210s, SEQ ID NO 256 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 256 ccgaatactic tictaaaac cc gat 23

<210s, SEQ ID NO 257 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 257 ccgt.cgc.ctt cotcc.gacga a 21

<210s, SEQ ID NO 258 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 258 tact actacg cc.gcttacgt cc 22

<210 SEQ ID NO 259 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 259 acgtaaaata aacaat caac tat cq 25

<210s, SEQ ID NO 260 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 260 talacac Coala accgaaaaac g 21

<210s, SEQ ID NO 261 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 261 aacgaatcca cataccc.ga 19

<210s, SEQ ID NO 262 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 9,371,569 B2 155 156 - Continued

<4 OOs, SEQUENCE: 262 cgct actic ct taaaaacgcc

<210s, SEQ ID NO 263 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 263 acaacgaaat cqaaaatcgt aa 22

<210s, SEQ ID NO 264 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 264 accatt cocq act cotcgt. 19

<210s, SEQ ID NO 265 &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct < 4 OO SEQUENCE: 265 gtttggttcg ggittagcgtt aatticggttt togtggaagt cqtggcgaaa ggcgagaggg 6 O gtaaaaagtt gagaaatagg cagcgggag agataagtag g

<210s, SEQ ID NO 266 &211s LENGTH: 110 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 266 ttgcgttitta tttgtattitc gcgtcgtttc gcggttcgcg gttgatt cqt ttitt.cggittt 6 O gcgggtttitt ggagttitt at tttittagagt cq9ttaagtic gttcgittaag 11 O

<210s, SEQ ID NO 267 &211s LENGTH: 114 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 267 ttitt attgcg agt catcggit cqttgttatg gacgtttatt at agttctggit gtcgtagagt 6 O cgggagggitt C9tcgtttitt taggg tattt titcggaggcg ataagttcgg tata 114

<210s, SEQ ID NO 268 &211s LENGTH: 106 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct

<4 OOs, SEQUENCE: 268 US 9,371,569 B2 157 158 - Continued tgtataggag ticgaagggac gitattacgtt agttt tagtt cq9ttittagc gatagittaac 6 O gtttitttgta gcgcggcggit titcgaagt cq t cqtt cqgag ttgttt 106

<210s, SEQ ID NO 269 &211s LENGTH: 95 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 269 gttaggtaag titacggcg agcgtaaggg aaggggittag titattgatta gcggtag taa 6 O ttgtaggaat citcgt.cgta gttgtagt cq tttitt 95

<210s, SEQ ID NO 270 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 270 atttaatgag gacgg taggit agc gaggttt tatt Caagt tttitcggcgt tatgagtagt 6 O taataggagt togtgtagaa gCagtttgt t 91

<210s, SEQ ID NO 271 &211s LENGTH: 117 & 212 TYPE DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 271 tittaatataa gtcgggittac gttcgagggit aataatatga ttaaaattat agtaggaatt 6 O ataataagga ataagattta ggittaaagta aatatagcga tagtttittgc gtcgitat 117

<210s, SEQ ID NO 272 &211s LENGTH: 116 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 272 ttagggagta agtgcgtttg cgc.gcggtgt gcgtttittaa acgcgattta aggcgt.cggg 6 O tttgttgtta attaattata agg tagtttc gttcgagcgt aggttaatcg gtttitt 116

<210s, SEQ ID NO 273 &211s LENGTH: 113 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct

<4 OOs, SEQUENCE: 273 tagacggitta cagtaggcg gtaggttcgt ttagggacg C9tttggitat cqcggcgttg 6 O tcqtttagga gcggttitt cq aagttittatt tttitcgt.cgt cqttittaaga agg 113

<210s, SEQ ID NO 274 &211s LENGTH: 107 &212s. TYPE: DNA US 9,371,569 B2 159 160 - Continued

<213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 274 tttitt.cgt at tittaggaagt ggcgcggittt gtcgagggta gcgtggagga ggalagaggag 6 O gcgcggittta acgcgatcga agttt cqtcg taalaggttcg ggaggaa 1. Of

<210s, SEQ ID NO 275 &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OO > SEQUENCE: 275 tittggggttc gattatattt cqgttagcgc gttittaggitt titcgatttitt tdtag taggit 6 O gttt citatic gcggcgittag ggat.cggttt C9gagttitt C g

<210s, SEQ ID NO 276 &211s LENGTH: 115 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 276 gttatttittc gg.cgggttcg ttttittttitt ttggtttitta gtttittattt tittatggtcg 6 O titcgggg.cgt ttt tagttgt ttaggittaga gaggtggcga agggg.cgatt attag 115

<210s, SEQ ID NO 277 &211s LENGTH: 113 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OO > SEQUENCE: 277 tacgcgtagg ttittaagt cq cqgttaatgg gcgacgcggit cqtagatt.cg titcggitttcg 6 O ttttgttttgttgagttttitt C9gtcgggitt gcggggtttic gtttagttcg gga 113

<210s, SEQ ID NO 278 &211s LENGTH: 95 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OO > SEQUENCE: 278 tittgatttitt gaaag.cgt.cg ttgcgttt C. c.gtcgcgggit agg tagggcg ggattitt tag 6 O gaggat.cggt agaggcgc.gt at aggtgcgt gatgt 95

<210s, SEQ ID NO 279 &211s LENGTH: 119 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct

<4 OO > SEQUENCE: 279 tttittaggga agtaaag.cgt. c9ttittctgtc. gtaggitatic agacgt.cgtt tagatggaag 6 O aaattittgga gatgcgcgtt ttt at atcgg tt.cgcggcg titcgggttta gtattacgt. 119 US 9,371,569 B2 161 162 - Continued

<210s, SEQ ID NO 280 &211s LENGTH: 101 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 280 tagtttggta gttagcgggit cqgggcgttt agttt tattt tttagagcgt toggttittg 6 O tgtttgaagg ttaaat agtt tacggitt at titcggaggitt t

<210s, SEQ ID NO 281 &211s LENGTH: 106 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 281 at agggggag titcggtacgg cqcgggcgtt taggagagaa ggaataataa atggatgagg 6 O gggatgttta gggttgttitt C9ggatagtic gagcggggta aattgt 106

<210s, SEQ ID NO 282 &211s LENGTH: 90 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 282 cgtttittata gggittittggit toggacgt.cgt cqtcqtcgtt gttatcgttt ttgatttaag 6 O ttatttitt cq t taggtgagt titcgagattit 9 O

<210s, SEQ ID NO 283 &211s LENGTH: 120 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 283 tagttgtatic ggtttaggcg ttittggtggg gtgggaagga titcgagt cqt atttgaatga 6 O aggittagttt ttttittaaga tattaattag gtagggagaa atcgaagggit acgaagg tag 12 O

<210s, SEQ ID NO 284 &211s LENGTH: 152 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct

<4 OOs, SEQUENCE: 284 gttatggcga tigcggttt cq gagagcgtac gtttgtc.gcg gtcgg tatgg aaacgttitt C 6 O gttaggttcg gggggg.tc.gt tattggit cq atttaataga cqcgggtggg tagtttagt c 12 O gtat cittaa gttcggtcgt tttittaggitt gg 152

<210s, SEQ ID NO 285 &211s LENGTH: 96 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: US 9,371,569 B2 163 164 - Continued <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 285 gaaggtagcg tttitt.cgatg gtgagtaggit tttgtaggac goggtcgttt C9gagtaggit 6 O tgcggttt cq tacggittittg cggat.cgggt ttattt 96

<210s, SEQ ID NO 286 &211s LENGTH: 92 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 286 aattittaggit tagagggitta t cqcgttt at gcgaggit cqg gtgggcgggit cottagttt C 6 O gttittgggga ggggttcgcg ttgttgattg gt 92

<210s, SEQ ID NO 287 &211s LENGTH: 94 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OO > SEQUENCE: 287 gaggggg tag gaaagt cqcg titcgttttitt attatttatt ttt tatttitt attattgggg 6 O ggttcggagc gcgcgaggitt tttaggtogt titcg 94

<210s, SEQ ID NO 288 &211s LENGTH: 91 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 288 gggacgattt titcgttgttc ggggttitt cq aacggcgggg gcgggaggcg gtaatttatt 6 O cggagcgcgt. C9gaga.gcga gagtagtaga a 91

<210s, SEQ ID NO 289 &211s LENGTH: 92 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 289 aattitcgttt gtagagtcgt. c9tcgt.cgt.c gtcgt.cggag gagcgagtc.g. atttitttittt 6 O tttittttitt c gaag.cgaagt ttittaatata ga 92

<210s, SEQ ID NO 290 &211s LENGTH: 82 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct

<4 OOs, SEQUENCE: 290 gtcggtaagg tttggaga.gc ggttgggttc gcgggatt.cg cgggtttgta titcgtttaga 6 O titcggacggg ttttgttatt tt 82 US 9,371,569 B2 165 166 - Continued

<210s, SEQ ID NO 291 &211s LENGTH: 90 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 291 ttagaagtaa tittaggcgcg titcgttggitt tttgagcgtt aggaaaagtt C9gagittaac 6 O gatcggtcgt t cqgtt attg tacggggttt 9 O

<210s, SEQ ID NO 292 &211s LENGTH: 108 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 292 aatttgattt gtgttgttgt at cqcgtttitta gcgattit.cgg atttattgcg ttgttagggg 6 O tittgggggtg ggtttitttgt tdtttittgcg acgatattitt tacgttt c 108

<210s, SEQ ID NO 293 &211s LENGTH: 103 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 293 gtttacgcga tttittgggac gtcggagata acggggttitt toggaaggcg cggagttcgg 6 O ggaagttctgg gatgtgcgcg tagt catgt togtagggitt ttt 103

<210s, SEQ ID NO 294 &211s LENGTH: 1.OO &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 294 agggitttitt C ggagtcgttt attagggittt tttgggggtt cq9tttcgat tdgg tagggg 6 O gatttggata gggttt ciga gcgtggagac ggttgaggaa

<210s, SEQ ID NO 295 &211s LENGTH: 103 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct <4 OOs, SEQUENCE: 295 agg.cgt.cgta tittatagogt tttgttcgcg tatatattitt ttittggggitt ggttgtaaat 6 O ttgt atgatt tacgtttaaa gaatgtc.gcg gaaagtalagg gga 103

<210s, SEQ ID NO 296 &211s LENGTH: 117 &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic construct

<4 OOs, SEQUENCE: 296