(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`‘
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`5 February 2009 (05.02.2009)
`
`(51) International Patent Classification:
`C12Q 1/68 (2006.01)
`
`
`
`—
`PCT
`
`(21) International Application Number:
`PCT/EP2008/006237
`
`(22) International Filing Date:
`
`29 July 2008 (29.07.2008)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/952,815
`
`English
`
`English
`
`30 July 2007 (30.07.2007)
`
`US
`
`(71)
`
`ROCHE DIAGNOSTICS
`Applicant (for DE only):
`116,
`68305
`GMBH [DE/DE]; Sandhofer Strasse
`Mannheim (DE).
`
`(71) Applicant(for all designated States except DE): ¥. HOFF-
`MAN-LA ROCHE AG[CH/CH]; Grenzacherstrasse 124,
`CH-4070 Basel (CH).
`
`(72)
`(75)
`
`Inventor; and
`Inventor/Applicant (for US only}: HIGUCHI, Russell
`[US/US]; 3258 Liberty Avenue, Alameda, CA 94501 (US).
`
`(10) International Publication Number
`WO 2009/015863 A2
`
`AO, AT, AU, AZ, BA, BB, BG, BIT, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
`EG,ES,FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
`IL, IN, IS, JP. KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LI, LU, LY, MA, MD, ME, MG, MK, MN, Mw,
`MX, MY, MZ, NA,NG,NI, NO, NZ, OM,PG,PH,PL, PT,
`RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ,
`‘TM, ‘IN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM,KE, 1.S, MW, MZ, NA, SD, SL, SZ, TZ, UG, 7M,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR,HR, HU,IE,IS, IT, LT, LU, LV, MC, MT, NL,
`NO,PL, PT, RO, SE, SL SK, TR), OAPI (BF, BJ, CF, CG,
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`
`as to the applicant’s entitlementto claim the priority of the
`earlier application (Rule 4.17(iii))
`of inventorship (Rule 4.17(iv))
`
`Published:
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`
`without international search report and to be republished
`upon receipt of that report
`
`(54) Title: METHODS OF IDENTIFICATION USING METHYLATION OF CPG
`
`ase
`rm.
`
`Pu’CG
`Py G
`
`---
`:
`
`
`
`Pu"CG
`Py G ———
`
`oo
`Enzymatically “blunt”
`ends of DNA;ligate ta
`“adapter” or “linker”
`synthetic dsDNA of
`known sequence
`
`{
`
`ot
`
`{
`
`PCRtargeting the
`“methylation-specific” junction
`sequence atsite ofligation —
`methylation-specific amplicon
`results. Nested-PCR increases
`specificity
`—» ————>
`
`
`<—
`
`: —
`Figure 1. ES
`
`(57) Abstract: The present inventionrelatesto the identification of methylated nucleotides in samples of genomic DNA.Thepresent
`invention also relates to methods of diagnosis of specific conditions by identification of specific methylated nucleotides.
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`[ti‘
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`WO2009/015863A2IIMITIMNIUINNITINIAIMINTITERAM
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`METHODSOF IDENTIFICATION USING METHYLATION OF CPG
`
`FIELD OF THE INVENTION
`
`The present inventionrelates to the identification of methylated nucleotides in samples of
`genomic DNA. Thepresentinventionalso relates to methods of diagnosis ofspecific
`conditions by identification of specific methylated nucleotides.
`
`BACKGROUNDOF THE INVENTION
`
`The detection of 5-methyl cytosine in human DNA,usually at CpG dinucleotides, is
`important diagnostically because the methylation at such cytosines, particularly at gene
`control sequences, (e.g. promoter sequences) is frequently associated with the onset of
`cancer. This so-called epigenetic (sinceit is not in the usual sense heritable) modification of
`DNAis also important in development and frequently results in gene silencing. In cancer,
`the epigenetic change is aberrant andcanresult in the silencing of genes involved in the
`suppression of tumor formation,or alternatively the activation of genes involved in
`
`oncogenesis.
`
`Current widely used methodsto detect such DNA modification use treatment of DNA with
`the chemicalbisulfite and have disadvantages with respect to performing a robust
`diagnostic assay. Amongthese are high complexity, the lengthy amountoftime required,
`lack of reproducibility and significant loss of the DNAto be detected.In addition, the use of
`bisulfite is incompatible with the use of uracil-n-glycosylase in the control of carryover PCR
`product contamination. There is need for a method without these disadvantages.
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`At the sametime there is a need for methods that detect such DNA modifications with high
`
`sensitivity and in the presence of high backgroundlevels of the same DNA sequence,
`unmodified. In a tumor, notall the cells contain DNAthat is methylated at the sequence of
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`interest — in fact, the majority of cells may not. Furthermore, in the case of early detection of
`cancer using either disseminated tumorcells or tumor DNA that can be found in the
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`bloodstream,the vast majority of DNAis not methylated at the sequenceofinterest. At
`most, only a small percent of copies of that sequence may be methylated. The concentration
`of such sequences maybeless than a single copy per milliliter of sample volume. The need
`for both highsensitivity and high specificity in detection is both clear anddifficult to obtain
`by previous methods.
`
`SUMMARYOF THE INVENTION
`
`Thepresent inventionis directed to a methodof detecting methylated DNAata specific
`locus in a sample, comprising:(a) treating the sample with a methyl-active cleavage method
`that cleaves DNAat a consistentsite in the presence of methylated DNAat the specific locus
`in the sample, (b) adding primers which compliment DNAat or nearthespecific locus,(c)
`subjecting the sample to a polymerase chain reaction and generating an amplification
`product whenthere is methylated DNAatthe specific locus in the sample, and (d) detecting
`the presenceof the amplification product, indicating the presence of methylated DNAat the
`specific locus in the sample.
`
`Preferred according to the present invention is a method of detecting methylated DNAat a
`specific locus in a sample, comprising: (a) treating the sample with a methyl-active method
`that results in base excision at a consistentsite in the presence of methylated DNA atthe
`specific locus in the sample,(b) altering the DNA sequenceatthe site of cleavage or base
`excision, (c) adding primers and/or probes which compliment DNAatornearthe specific
`locus andatleast one of whichis capable of specifically recognizing the altered DNA
`sequence, (d) subjecting the sample to a polymerasechain reaction and generating an
`amplification product and/or probe signal when there is methylated DNAat the specific
`locus in the sample, and (e) when amplification product or probesignalis specifically
`generated, detecting the presence of the amplification product, indicating the presence of
`methylated DNAatthe specific locus in the sample.
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`Thepresent invention further comprises methods of diagnosing certain conditionsby the
`detection of methylated cytosineat a specific locus in genomic DNA samples.
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`Theinventionis further directed to methods of detecting methylated DNAat a specific
`locus in a sample, comprising treating the sample with a methyl-active cleavage methodthat
`cleaves DNAata consistentsite in the presence of methylated DNAatthe specific locus in
`the sample, adding primers which compliment DNAator near the specific locus, subjecting
`the sample to a polymerase chain reaction and generating an amplification product when
`there is methylated DNAatthe specific locus in the sample, and detecting the presence of
`the amplification product, indicating the presence of methylated DNAat the specific locus
`in the sample.
`
`In particular embodiments, the sample comprises genomic DNA.
`
`In preferred embodiments,the specific locus is a promoter region of a knowngene. In
`further embodiments, the sample comes from a patient, and the presence of methylated
`DNAat the promoterregion of the knowngeneindicates the presence cancerouscells in the
`patient.
`
`In further preferred embodiments, the methyl-active cleavage methodthat cleaves DNAat a
`consistent site in the presence of methylated DNAis a methyl-active restriction enzyme. In
`further embodiments, the methyl-active cleavage method that cleaves DNAat a consistent
`site in the presence of methylated DNAis treatment with 5-methyl deoxycytidine
`glycosylase/lyase, treatment with 5-methyl deoxycytidine glycosylase followed by a separate
`apurinic/apyrimidinic lyase (or separate apurinic/apyrimidinic endonuclease) or treatment
`with 5-methyl deoxycytidine glycosylase followed byalkaline hydrolysis.
`
`Thepresentinvention further comprises methodsof detecting cancerin a patient by
`detecting methylated DNAata specific locus in a sample from thepatient, comprising
`treating the sample with a methyl-active cleavage method that cleaves DNAat a consistent
`site in the presence of methylated DNAatthe specific locus in the sample, adding primers
`which compliment DNAatornearthespecific locus, subjecting the sample to a polymerase
`chain reaction and generating an amplification product whenthere is methylated DNA at
`the specific locus in the sample, detecting the presence of the amplification product,
`indicating the presence of methylated DNAatthespecific locusin the sample, and detecting
`cancerin the patient from the presenceof the amplification product.
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`In certain embodiments, the sample comprises genomic DNA. In certain embodiments,the
`specific locus is a promoterregion of a knowngene.In certain embodiments,the methyl-
`active cleavage methodthat cleaves DNAata consistentsite in the presence of methylated
`DNAis a methyl-active restriction enzyme.In certain embodiments, the methyl-active
`cleavage methodthat cleaves DNAata consistentsite in the presence of methylated DNAis
`treatment with 5-methyl deoxycytidine glycosylase/lyase, treatment with 5-methyl
`deoxycytidine glycosylase followed by a separate apurinic/apyrimidinic lyase (or separate
`apurinic/apyrimidinic endonuclease) or treatment with 5-methyl deoxycytidine glycosylase
`followed by alkaline hydrolysis.
`
`In certain embodiments, the present invention comprises methods ofdetecting cancerin a
`10
`patient by detecting methylated DNAatspecific locus in genomic DNAinasample from
`the patient comprising treating the genomic DNA with a methyl-active restriction enzyme
`to create a cleavage product, adding primers which compliment DNAatornear the specific
`locus, subjecting the sample to a polymerasechain reaction to obtain an amplification
`product when there is methylated DNAatthespecific locus, detecting the presence of the
`amplification product whichindicates the presence of methylated DNAat the specific locus,
`and detecting cancerin a patient by detecting the presence of the amplification product.
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`In preferred embodiments ofthe invention,the specific locus is a promoter region of a
`knowngene, the methyl-active restriction enzymeis E. coli McrBC and/or the method
`further comprises the steps of creating blunt ends on the cleavage product, ligating the ends
`of the cleavage productto create a closed circle of the cleavage product, andthe primers are
`oriented such that the amplification productcan only result from a closedligated circle of
`the cleavage product.
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`In certain embodiments,the present invention comprises methodsof detecting cancer in a
`patient by detecting methylated DNAatspecific locus in genomic DNAin a sample from
`the patient comprising treating the genomic DNA with a methyl-active restriction enzyme
`to create a cleavage product, creating blunt ends on the cleavage product, ligating the ends
`of the cleavage productto create a closed circle of the cleavage product, adding primers
`which compliment DNAatornearthespecific locus, wherein the primers are oriented such
`that an amplification product can only result fromaclosedligated circle of the cleavage
`product, subjecting the sample to a polymerase chain reaction to obtain an amplification
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`product, detecting the presence of the amplification product which indicates the presenceof
`methylated DNAatthe specific locus, and detecting cancerinapatient by detecting the
`presence of the amplification product.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Definitions
`
`The term “amplicon”refers to a double-stranded DNA molecule generated as the result of
`an amplification reaction, such as the Polymerase Chain Reaction.
`
`As usedin the presentinvention, the term “CpGsite” refers to the cytosine and guanisine
`dinucleotide which may be methylated at the cytosine in some genomic DNA molecules.
`Typically, the CpG dinucleotideis present within a larger nucleic acid sequence.
`
`The phrase “methyl-active restriction enzyme”in the present inventionrefers to a
`restriction enzyme whichonly cleaves DNA when methylated cytosine is presentin the
`DNA. Different such enzymes may require the methylated cytosine to be at a specific site.
`
`The phrase “methyl-active cleavage” in the present inventionrefers to a cleavage of nucleic
`acid whichoccursonlyin the presence of a methylated nucleic acid. In the present
`invention, methods of methyl-active cleavage include, but are not limitedto, the use of
`methyl-active restriction enzymes.
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`In the presentinvention, the term “5-methyl deoxycytidine glycosylase/lyase” refers to an
`enzyme,both a glycosylase anda lyase, whichis active in the presence of a 5-methyl
`deoxycytidine (Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marin MI, Martinez-
`Macias MI, Ariza RR, Roldan-Arjona T. Proc Natl Acad Sci US A. (2006) 103(18):6853-8;
`Gehring M, HuhJH, Hsieh TF, PentermanJ, Choi Y, HaradaJJ, Goldberg RB, Fischer RL.
`Cell. (2006) 124(3):495-506) . The glycosylaseactivity of the 5-methyl deoxycytidine
`glycosylase/lyase typically breaks the N-glycosidic bond between 5-methyl deoxycytidine
`and ribose of DNA.Thelyase activity, also knownas apurinic/apyrimidinic (AP) lyase,
`cleaves the DNA backbone3’ to the abasic sugar by a beta-elimination reaction.
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`Nucleic acid - In the presentinvention, the phrase “nucleic acid” mayrefer to any natural or
`synthetic nucleic acid, including, but not limited to, single-stranded and double-stranded
`nucleic acids, DNA, RNA, zDNA,synthetic nucleotide analogs, and peptide-linked synthetic
`
`nucleotides.
`
`GeneofInterest — In the present invention, the term “geneof interest” mayrefer to any
`coding or non-codingregion present in a genomic sequence thata researcherorclinician
`examines for methylation.
`
`Promoter region — In the present invention, a “control region” maybe any portion of a
`nucleic acid near a geneofinterest that is not necessarily included within the gene. A
`controlling region may or may nothave a direct regulatory effect on the expression of the
`gene ofinterest. A controlling regionis typically a region which may have a methylated 5-
`deoxycytidine in certain cells where the expression ofthe geneofinterestis affected.
`
`Methyl-Active Cleavage
`
`The present invention encompasses several methods for methyl-active cleavage. Any
`methodthat cleaves DNAin the presence of methylated CpG, but does not cleave DNA in
`the absence of methylated DNAcan beusedin thepresent invention. Methodsinclude, but
`are notlimited to, methyl-active restriction enzymes, such as McrBC (Stewart,F.J. and
`Raleigh E. A. (1998) Biol. Chem. 379: 611-616.) and 5-methylcytidine glycosylase combined
`with lyase.
`
`Amplification Methods
`
`A variety of amplification methodsare envisioned by the presentinvention, including, but
`notlimited to, the polymerase chain reaction (PCR), ligase chain reaction,androllingcircle
`replication.
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`Detection Methods
`
`Several methodsof detecting specific species of amplification product are consideredby the
`present invention. Methodsofdetection include, but are not limited to, incorporation and
`detection of labels, probe capture methods, Taqmanassays,electrophoretic methods, and
`hybridization methods. Labels include, but are not limited to, radio-labelled nucleotides,
`fluorophores, quantum dots, biotin-conjugated nucleotides, and chromogenic enzymes.
`
`A variety of methods maybe used which couple detection of methylated CpG ata specific
`locus and amplification. For example, a genomic sample maybe cleaved through a methyl-
`active enzyme whichis active in the presence of methylated CpGs. A segment of DNA
`resulting from such cleavage may be subjected to enzymatic “blunting”ofthe ends, to
`which an oligonucleotide adapter of a known sequenceis ligated. The sample may then
`have two primers added, one of which hybridize to a region nearthe specific locus, and the
`second ofwhich hybridizes to the known sequence onthe oligonucleotide adapter. The
`aforementioned exampleis illustrated below in Figure1.
`
`In preferred embodiments, the invention may be practiced as follows: A specifically
`detectable DNA sequencealteration may be produced enzymatically near the sites of two 5-
`methyl cytosines in DNA. As diagrammedin Figures 1 and 2 below,
`therestriction enzyme
`E. coli McrBC mayspecifically recognize and cleave dsDNAnear 5-methyl dC residuesthat
`are preceded directly by dA or dG (purines = Pu). These include 5-methyl dC’s that precede
`dG residues to form CpG dinucleotides, the majorsite of methylation in mammalian DNA.
`If the dC residue is not methylated, no cleavage takes place. For this reason McrBCis
`described as “methyl-active,” as opposed to “methylation sensitive,” which describes a
`larger knownclass ofrestriction enzymes for which the opposite is true, thatis, an
`unmethylated target sequence is cleaved while a methylated target sequenceis not.
`
`Strictly speaking, to cleave DNA, McrBC typically requires two Pu"Csites spaced from 55
`bp to up to 3 kbp apart. The cleaveage site would be approximate 30 bp from oneofthe two
`Pu"Csites. Because sites of DNA methylation involved in gene regulation contain a high
`density of methylated CpG dinucleotides, the situation diagrammed in Figures | and 2 that
`results in the production of a novel methylation-specific DNA fragment would likely occur.
`Note that the cleavages do not need to be between the recognitionsites, but are
`diagrammedthat way for example. Whenit does occur,the creation of a methylation-
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`specific sequence alteration at the cleavage site(s) is possible. For example, in both Figures 1
`and 2 below, the methylation-specific DNA fragmentis treated as described in the
`references to create ligatable fragmentends. If, as in Figure 1, the DNA fragmentis then
`allowed to cyclize in the presence of DNAligase, a covalently-closed DNA circle may be
`created. Theligated junction site and sequence may be determined empirically using
`samples containing methylated target genes from, for example, DNA from human tumors.
`This may be done using DNA cloning and sequencing. Once known,specific DNA primers
`may be designed and synthesized for PCR that would amplify a specific DNA productas
`diagrammed, with one primerspecific for the novel junction sequence. Amplification
`following cyclization of a DNA fragmentis referred to as “inverse” PCR (Ochman H,
`GerberAS, Hartl DL. Genetics. (1988) 120(3): 621-623.). Alternatively, as in Figure 2, a
`synthetic, dsDNA fragment knownasa “linker” or as an “adapter” can beligated to either
`or both ofthe fragmentends. Sinceall the sequences shownwill be known,a primerfor
`PCRcan be designedthat specifically targets the junction sequence between the
`methylation-specific fragment andthe linker. To increase specificity (as manydifferent
`fragments will also now contain the linker sequence), “nested” PCR may be done as
`diagrammed.
`
`It wouldalso be possible to target the novel junction sequences with fluorescent
`oligonucleotide probes compatible with PCR (e.g., Taqman probes), and use flanking PCR
`amplification primers. However, targeting the novel sequence with a PCR primerinstead
`of a probe has the advantagein the situation where the methylation-specific targetis a
`minority sequencein the backgroundofan amplifiable alternative (whichislikely if the
`diagnostic applicationis early cancer detection from DNAfoundin bodilyfluids such as
`serum or plasma — the majority of the DNAis “wildtype” and not methylated). If both are
`amplified, the signal that can be generated by the probe is reduced.If only the methylation-
`specific target is amplified, signal-to-backgroundis enhanced.
`
`With flanking primers, and using inverse PCR,it is possible that there may not be an
`amplifiable alternative.
`In the absenceof significant methylation in the region ofinterest
`the cleaved fragmentwill be large and the amplification primers may be placed suchthat, in
`the absence of methylation, the amplification product will be too large to amplify efficiently.
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`Other nucleic acid amplification methods, such as SDA, should be adaptableto detect the
`methylation-specific sequence changeas well.
`
`In other preferred embodiments,the invention maybe practiced as follows. As diagrammed
`in Figure 3, the enzyme 5-methy] deoxycytidine glycosylase/lyase can specifically recognize
`and remove a 5-methyl cytosine nucleotide from dsDNA(in particular from a CpG
`dinucleotide), leaving a one nucleotide gap thatis preceded 5’ by a nucleotide bearing a 3’
`
`phosphateor an a,B-unsaturated aldehyde (not shown), both of which can be removed
`using E.coli endonucleaseIV,leaving a free 3’ hydroxyl groupsuitable for nucleotide chain
`extension by a DNA polymerase. In Figure 3A, such a chain extension is performed using a
`single nucleotide triphosphate (dNTP) that is “mutagenic”. That is to say, the dNTP can be
`insertedefficiently at this position and, when the DNAstrand containing the mutagenic
`nucleotide is subsequently copied by DNA polymerase, a nucleotide is inserted that alters
`the original base sequence. An example, and notthe only example, ofthis is 5-
`bromodeoxyuracil nucleotide triphosphate (5-BrdUTP) which canbe efficiently inserted
`opposite a dG residue by a DNA polymerase, particularly in the absence of dCTP. But when
`copied in the presenceofall four natural dNTPs, a dA residue, rather than a dCresidue,is
`preferentially inserted (Lasken RS, Goodman MF.J Biol Chem. (1984) 259(18): 11491-5).
`To prevent an analog from being removed once incorporated, the polymerase canlack 3’
`exonuclease proofreadingactivity. Once such ananalogis incorporated,it can be ligated to
`the 5’ phosphateat the gap. Alternatively, the dNTP analog can be removed andchain
`extension from the 3’ OHofthe incorporated nucleotide analog can be made to proceed
`with DNA polymerase (lacking 3’ exonuclease) and the four natural dNTPs along with
`strand-displacementofthe preexisting, annealed DNAstrand.
`
`This is followed bythe separation of the two DNAstrandsby, for example, heat
`denaturation, and the primed synthesis by a DNA polymerase ofcopies of the strand
`containing the mutagenic nucleotide analog. Because of the analog, copies are made that
`havean altered nucleotide base sequence. Once a genomic methylation site and
`surrounding sequenceis identified, model experiments can be performedtoidentify the
`specific sequence alterations produced by this procedure. Such an altered sequence can be
`efficiently andsensitively detected by primer-directed DNA amplification (e.g., PCR). PCR
`is well-knownto discriminate against single-base mismatchesto a primer,in particular
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`against mismatchesat the 3’ terminusof the primer; such mismatchescan be designed into
`a primer madefor a givenassay.
`
`Alternatively, as diagrammedin Figure 3B, a DNA polymerase (lacking 3’ exonuclease
`proofreadingactivity) and dGTP, dATP or dTTPalone can be provided. In the absence of
`dCTP a mismatched nucleotide base can be forced to incorporate opposite dG. This can be
`promoted by the presence of Mn™andtheuse of misincorporation prone polymerases
`such asviral reverse transcriptases. The single dNTP provided should not be the one
`expected to incorporate one base upstream of the 5-methyl dC replacement, as a nick or gap
`at this position mighttranslate to the replacementsite, giving a false positive result. Efficient,
`enzyme-mediated ligation of a nucleotide base mismatched to dG canalso be madeto take
`place (Lu J, Tong J, Feng H, Huang J, Afonso CL, Rock DL, Barany F, Cao W. Biochim
`Biophys Acta. (2004) 1701(1-2): 37-48.
`). Or, instead ofligation taking place, once
`misincorporation has been allowed to take place, the remaining three natural dNTPscan be
`provided and,in the absenceof 3’ exonuclease proofreading activity, efficient extension
`from the incorporated, mismatched nucleotide can be madeto occur. As above,this results
`in a specifically knowable, amplifiable and detectable sequence change.
`
`BRIEF DESCRIPTION OF THE FIGURES
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`Figure 1 illustrates a method of amplification detection. Theillustrated method employs
`the ligation ofa specific linker, and the use of primers which hybridize to the linker and the
`excised DNA. Nested primers within the sequence of the excised DNAincrease the
`specificity of the amplification and detection.
`
`Figure 2 illustrates another methodof amplification anddetection using “inverse” PCR.
`Followingexcision, the excised DNA fragmentis cyclized, and primers which amplifyan
`amplicon over the juncture ofligation are employed to amplify and detect successful
`cleavage of the DNA.
`
`Figure 3 illustrates another methodof amplification and detection using nucleotide excision
`and mutagenesis. Using a glycosylase specific to 5-methy]cytidine, nucleotide excision is
`initiated. The excised nucleotide can be replaced by a mutagenic analog (A), or by a
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`misincorporated natural nucleotide (B). In either case, copying by DNA polymeraseofthe
`modified DNAstrandresults in a sequencealteration that is specifically amplifiable and
`detectable by, for example, PCR.
`
`EXAMPLES
`
`The following are prophetic, and do not represent actual experiments.
`
`Example 1
`
`A sample offluid may be taken from a patient. Genomic DNA maybeextracted from the
`patient sample using known methods.
`
`The genomic DNAextractedis then treated with a restriction enzymethatis active in the
`presence of methylated cytosine in CpGsites, yielding an enzyme-treated sample. The
`enzyme-treated sampleis then combined with a linker whichis ligated to the ends of
`molecules cleaved by the restriction enzyme.
`
`The mixture is combined with a primer which hybridizes to a sequence near a promoter
`region of a geneofinterest, and a second primer which hybridizesto the linker which is
`ligated to the ends of the molecules cleaved by therestriction enzyme. Using the primer
`and the second primer, the mixture may then be subjected to an amplification reaction,
`such as PCR. Whenspecific CpGsites are methylated, a genomic DNA moleculeis cleaved,
`a linkeris ligated, and a specific amplicon is generated from the amplification reaction. In
`the absenceofthe specific methylated CpG,the specific ampliconis not generated.
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`Thespecific amplicon maythen be detected througha variety of known means.If the
`specific amplicon is detected, indicating the methylatedstate of the specific CpGsite, then a
`specific neoplasmic state maybe indicated and diagnosed.
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`Example 2
`
`In a further example, a solid tumor biopsy maybe obtained fromapatient. Established
`techniques maybe used to extract genomic DNAfrom the solid tumorbiopsy to yield a
`sample.
`
`The genomic DNA sampleis then treated with a restriction enzymethatis active in the
`presenceof methylated cytosine in CpGsites, yielding an enzyme-treated sample. The
`enzyme-treated sample maybethentreated in a matter which creates blunt-ended double-
`stranded DNA. This sample may henbe subsequently treated withaligase, creating circular
`DNAfrom short pieces of DNA. This exampleis illustrated below in Figure 2.
`
`The sample may then be combinedwitha set of primers which hybridize to sequences near
`a genomic sequenceofinterest which, whencertain sites near a genomic sequence of
`interest are cleaved andligated into a certain circular DNA,generate an amplicon only
`possible when thecertain circular DNAis present. The sample andset of primers may be
`subjected to an amplification reaction, generating a specific amplicon when CpGsites near
`the genomic sequenceof interest are methylated.
`
`Thespecific amplicon maybedetected, indicating that the solid tumor from which the
`biopsy was taken wasofa specific type of tumorindicated by the methylation of CpGsites
`near the genomic sequenceofinterest.
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`20
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`Example 3
`
`Ina further example, a solid tumor biopsy may be obtained from a patient. Established
`techniques maybe used to extract genomic DNA from the solid tumorbiopsy toyield a
`sample.
`
`25
`
`The genomic DNA sampleis then treated with a combined 5-methylcytosine
`glycosylase/lyase thatis active in the presence of methylated cytosine in CpGsites or a 5-
`methylcytosine glycosylase thatis active in the presence of methylated cytosine in CpGsites
`followed by a separate AP lyase or AP endonuclease,yielding an enzyme-treated sample
`with gaps in its double stranded DNA. The enzyme-treated sample maybethen treated in a
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`mannerthat allowsthe gaps to befilled by a nucleotide with different base-pairing
`specificity than dC.
`
`The sample is combinedwith a primer which hybridizes to a sequence in a cancer-related
`gene ofinterest, and a second primer which hybridizes specifically to a now mutated
`sequencenearthe first primer. In certain embodiments,for the greatest specificity in PCR,
`the 3’ nucleotide or the 3’ penultimate nucleotide of the second primeris opposite the
`mutated base. Using the primer and the second primer, the mixture may then be subjected
`to an amplification reaction, such as PCR. Whenspecific CpGsites are methylated, a
`specific ampliconis generated from the amplification reaction. In the absence ofthe
`specific methylated CpG,the specific amplicon is not generated.
`
`The specific amplicon maythen be detected through a variety of known means.If the
`specific ampliconis detected, indicating the methylated state of the specific CpG site, then a
`specific neoplasmic state may be indicated and diagnosed.
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`CLAIMS
`
`A methodof detecting methylated DNAata specific locus in a sample, comprising:
`
`treating the sample with a methyl-active cleavage method that cleaves DNA ata
`consistentsite in the presence of methylated DNAatthe specific locus in the sample,
`
`adding primers which compliment DNA ator nearthe specific locus,
`
`subjecting the sample to a polymerase chain reaction and generating an amplification
`product whenthereis a successful methyl-active cleavage, but no amplification when
`there is no successful cleavage, and
`
`detecting the presence of the amplification product,indicating the presence of
`methylated DNAatthe specific locus in the sample.
`
`The methodofclaim 1, wherein the specific locus is a promoter region of a known
`
`gene.
`
`The methodofclaim 2, wherein the sample comes from a patient, and wherein the
`presence of methylated DNAat the promoterregion of the knowngene indicates the
`presence cancerouscells in the patient.
`
`The methodofclaim 1, wherein the methyl-active cleavage method that cleaves DNA
`at a consistentsite in the presence of methylated DNAis a methyl-active restriction
`
`enzyme.
`
`20
`
`25
`
`The methodofclaim 1, wherein the methyl-active cleavage methodthat cleaves DNA
`at a consistentsite in the presence of methylated DNAis treatment with 5-methyl
`deoxycytidine glycosylase/lyase.
`
`A methodof detecting cancer in a patient by detecting methylated DNAata specific
`locus in a sample from the patient, comprising:
`
`treating the sample with a methyl-active cleavage methodthat cleaves DNAat a
`consistent site in the presence of methylated DNAatthe specific locus in the sample,
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`adding primers which compliment DNAator nearthespecific locus,
`
`subjecting the sample to a polymerasechain reaction and generating an amplification
`product whenthere is methylated DNAatthe specific locus in the sample,
`
`detecting the presence of the amplification product, indicating the presence of
`methylated DNAat the specific locus in the sample, and
`
`5
`
`detecting cancerin the patient from the presence of the amplification product.
`
`The methodofclaim 1 or 6, wherein the sample comprises genomic DNA.
`
`The methodofclaim 6, wherein thespecific locus is a promoter region of