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WO2009115313A1 - Procédé et composition pour analyse de méthylation - Google Patents

Procédé et composition pour analyse de méthylation Download PDF

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Publication number
WO2009115313A1
WO2009115313A1 PCT/EP2009/002003 EP2009002003W WO2009115313A1 WO 2009115313 A1 WO2009115313 A1 WO 2009115313A1 EP 2009002003 W EP2009002003 W EP 2009002003W WO 2009115313 A1 WO2009115313 A1 WO 2009115313A1
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dna
methylation
cytosine
probes
promoters
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Tamara Maes
Olga Duranny-Turk
Elena Aibar-Duran
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Oryzon Genomics SA
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Oryzon Genomics SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the invention relates generally to biotechnology and, more particularly, to methods and compositions for correlating the methylation status of DNA with phenotypic information.
  • DNA methylation is a well-known epigenetic modification affecting gene regulation (e.g., gene expression). Cytosine methylation is characterized by the presence of a methyl group on the carbon-5 position of cytosine (referred to as 5-methylcytosine). This modification is responsible for an important form of gene regulation in eukaryotes. Most studies on DNA methylation have focused on the methylation status of the cytosine bases included in the CpG dinucleotide, which seems to be the preferential site for the modification.
  • CpG dinucleotides In the mammalian genome there are about 50 million CpG dinucleotides. In mammals, cytosine methylation occurs almost exclusively at CpG dinucleotides, which are underrepresented in the genome with the exception of CpG islands; regions of the genome containing a high percentage of CpG dinucleotides and are approximately about 1 kb in size. These small CpG-rich regions, in many cases, are associated with promoter regions. Cytosine methylation results in transcriptional repression either by interfering with transcriptional factor binding or by inducing a repressive chromatin structure.
  • DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation. Alterations in DNA methylation are associated with many human diseases and are a hallmark of cancer. A decrease in the total amount of cytosine methylation is observed in many human neoplastic tissues and as indicative of global desregulation. At the same time, aberrant promoter hypermethylation has been observed in sporadic cancer and is thought to contribute to carcinogenesis by inactivating tumor-suppressor genes. Moreover, the change in the methylation state of regulatory genes (hypomethylation or hypermethylation), being a primary event, is frequently associated with the neoplastic process and is proportional to the severity of the disease (J. Paluszczak and W. Baer-Dubowska (2006) J. Appl Genet. 47(4):365-75).
  • the bisulphite methodologies are based on treating the sample with bisulfite under appropriate conditions.
  • Bisulphite treatment leads to the conversion (deamination) of cytosine to uracil, whereas methylcytosine is not converted to uracil under these conditions.
  • Amplification of the bisulphite treated DNA with strand-specific primers results in the uracil being replaced by thymine (the C/G pair is converted to a T/A pair), thus distinguishing cytosine and methylcytosines.
  • the subsequent cloning and sequencing of the PCR products allows the methylation percentage at any cytosine base in the region to be analyzed.
  • the methylation sensitive method involves digestion of DNA with a methylation-sensitive restriction endonuclease, which fails to cut if a cytosine base in the recognition sequence is methylated; the endonuclease most commonly used in this kind of analysis is Hpall, which recognizes the CCGG sequence and fails to cut if either of the cytosines is methylated.
  • Hpall the endonuclease most commonly used in this kind of analysis
  • a subsequent PCR amplification will yield a product only if the DNA is not cut, i.e., if the cytosines are methylated.
  • Methyl-binding proteins and anti-methylcytosine antibodies methods are both immunocapturing approaches to enriching methylated DNA. Methylated enriched DNA is then amplified and employed on massive screening assays as microarrays.
  • the main limitations of these approaches are that immunoprecipitation of large fragments increases the likelihood of co-precipitating adjacent, unmethylated DNA, causing false-positive signals, especially on high-density oligonucleotides arrays.
  • these approaches require relatively large amounts of input DNA (2-20 ⁇ g); making it very difficult to employ these methods in clinical research.
  • Bisulfite treatment of genomic DNA, followed by genomic sequencing is a more informative methodology, since it assesses the methylation status (expressed as methylation percentage) of all the cytosine bases (both CpG and non-CpG) in a given sequence, whereas restriction endonuclease methods yield information exclusively on the methylation status of the cytosine bases in the recognition sequence(s).
  • Bisulfite treatment has the disadvantage that the processing of the samples is both more complex, particularly in the DNA modification and purification phases, and time consuming, owing to the number of clones that need to be prepared for sequencing; the disadvantage of the latter is that it may be necessary to use different endonucleases to investigate different cytosine bases, thus increasing the number of the samples that need to be amplified by PCR. Therefore, despite being less exhaustive than the bisulfite technique, Hpall/PCR may still be considered useful because it is an easy technique for studying DNA methylation.
  • U.S. Patent 6,214,556 describes genomic DNA PCR amplification following bisulphite treatment, using either degenerate oligonucleotides or oligonucleotides which are complementary to adaptors ligated to the ends of cleaved DNA, and final PCR amplification of DNA material followed by hybridization on a microarray.
  • This strategy typically results in the loss of information since there is difficulty on specific-promoter-sequence amplification due to high CG content, which can lead to promoter information loss during genome-wide application, therefore compromising initial sample fidelity.
  • Biochem., 369:120-127 describe a method for methylation analysis involving shearing genomic DNA by vortex and/or syringe, treatment with bisulphite, PCR amplification with degenerate primers pairs, and detection by methylation specific PCR.
  • PCR based amplification of high GC content regions of genomes have proven problematic (McDowell et al., Nucleic Acid Research (1998) 26:3340-3347). PCR Amplification of high GC rich regions give weak signals when amplified using standard PCR conditions and the amplifications can result in non-specific amplification.
  • Various additives and enhancing agents that typically alter melting temperatures have been proposed like DMSO, betaine, formamide, glycerol, non-ionic detergents, and the like to increase PCR efficiency, specificity, and reproducibility (Musso et al. (2008) J. MoI. Diag. 8:544-550). It is related in Sahdev et al.
  • Mamedov et al. ((2008) Comp. Biol. & Chem. 32:452-457) present a theoretical analysis of how high GC rich regions hinder PCR amplifications and suggest that shorter annealing times can aid in more efficient PCR amplification.
  • the method is useful for characterizing the methylation status or methylation profile of DNA.
  • the method can be used to examine the methylation status/profile of a specific promoter/CpG island and/or a plurality of promoters/CpG islands.
  • the compositions of the invention can be used for assessing the DNA methylation of genomic DNA.
  • the method is useful in numerous applications including the diagnosis and prognosis of diseases having altered DNA methylation patterns.
  • the method of the invention is also useful for biomarker discovery.
  • the invention can be used to identify specific biomarkers associated with phenotypes and for establishing methylation fingerprints (e.g., patterns, status, profiles, or the methylome).
  • Methylation patterns, status, profiles, and the methylome as determined by the methods of the invention can be associated with phenotypes (prognosis, diagnosis, response to therapeutics, etc.).
  • the methods and compositions of the invention are useful for determining genome-wide methylation patterns.
  • methods of the invention involve determining the methylation of DNA by treating the DNA with an agent capable of distinguishing cytosine and 5-methylcytosine, subjecting the resulting DNA to in vitro transcription, and detecting the products of the in vitro transcription to determine the methylation of the DNA.
  • the methods can comprise comparing treated DNA (according to the methods of the invention) versus untreated DNA or simply determining the profile of the treated DNA.
  • the method of the invention comprises:
  • detecting the RNA to determine the methylation of the DNA comprises determining the sequence of the RNA, or a nucleic acid derived from the RNA, to determine the identity of the sequences obtained from the in vitro transcription, thereby determining the methylation of the DNA.
  • detecting the RNA to determine the methylation of the DNA comprises hybridizing the RNA, or a nucleic acid derived from the RNA, to a microarray to determine the identity of the sequence(s) obtained from the in vitro transcription, thereby determining the methylation of the DNA.
  • the methylation of 50 or more cytosines in the DNA sample is determined.
  • the methylation of from 50 to 100,000 cytosines in the DNA sample is determined.
  • the methylation of from 100 to 100,000 cytosines in the DNA sample is determined.
  • methylation of three or more promoters in the DNA sample is determined.
  • the methylation of six or more promoters in the DNA sample is determined. In some aspects of the invention, the methylation of from three to 10,000 promoters in the DNA sample is determined.
  • the methylation of six to 10,000 promoters in the DNA sample is determined.
  • the methylation of three or more CpG islands in the DNA sample is determined.
  • the methylation of six or more CpG islands in the DNA sample is determined.
  • the methylation of from three to 10,000 CpG islands in the DNA sample is determined. In some aspects of the invention, the methylation of from six to 10,000 CpG islands in the DNA sample is determined.
  • the DNA is not amplified prior to treatment with an agent that is capable of distinguishing cytosine from 5-methylcytosine.
  • treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine comprises bisulphite treatment.
  • treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine comprises fragmentation of the DNA, ligation of adaptors to the fragmented DNA, and treatment of adaptor ligated fragmented DNA with bisulphite.
  • in vitro transcription to synthesize RNA comprises synthesizing double-stranded DNA from the DNA obtained by treatment with an agent that is capable of distinguishing 5-methylcytosine from cytosine and transcribing the double-stranded DNA into RNA with a polymerase.
  • fragmentation of the DNA comprises treating the DNA with one or more restriction enzymes.
  • the one or more restriction enzymes are methylation insensitive.
  • the DNA is not amplified prior to treatment with an agent that an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the method is for diagnosing cancer.
  • the method is for prenatal diagnosis.
  • the invention provides a kit having (1) a component for fragmenting DNA, (2) a component for distinguishing 5-methylcytosine and cytosine, (3) an in vitro transcription component, and (4) instructions for using the components of the kit.
  • the invention provides a kit having component for distinguishing 5-methylcytosine from cytosine comprises reagents for selective bisulphate-mediated deamination of cytosine as compared to 5-methylcytosine.
  • the invention provides a kit where in vitro transcription component comprises adaptors.
  • the invention provides a set of probes for detecting for diagnosing cancer wherein said probes are designed to the methylation status of DNA treated with bisulphite and subsequent in vitro transcription.
  • the set of probes comprise probes for from two to 1000 promoters or CpG islands.
  • the set of probes comprise probes for from ten to 1000 promoters or CpG islands.
  • the set of probes comprise probes for from twenty to 1000 promoters or CpG islands.
  • the method of the invention involves (1) providing a sample of DNA, (2) treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine in the DNA and converting the resulting single-stranded DNA into double-stranded DNA, (3) subjecting the resulting double-stranded DNA to in vitro transcription, and (4) detecting the products of the in vitro transcription to determine the methylation of the DNA.
  • the sample of DNA for use in the invention can be from any source and/or organism.
  • the DNA can be from human cells, human cancer cells, circulating DNA, fetal DNA (isolated e.g., from maternal plasma), cancer cell lines, mammalian cells, mammalian cancer cells, mouse cells, cancer cells obtained from mice, plant cells, etc.
  • the sample of DNA can also be obtained by various methods, e.g., from a biopsy, blood sample, aspirate, tissue section, a fluid sample, swab, etc.
  • the step of treating DNA with an agent that distinguishes cytosine from 5-methylcytosine involves contacting the DNA with an agent that modifies either cytosine or 5-methylcytosine.
  • an agent that modifies either cytosine or 5-methylcytosine is alkaline bisulphite treatment under conditions which convert cytosine to uracil, but leaves 5-methylcytosine as 5-methyl cytosine.
  • the DNA can be contacted with an agent that modifies both cytosine and 5-methylcytosine, rendering them distinct. This step allows the later determination, at cytosine positions in DNA, whether it is 5-methylcytosine or cytosine.
  • the step involving treatment with bisulphite results in single-stranded DNA.
  • the single-stranded DNA can be converted to double-stranded DNA for the subsequent in vitro transcription step.
  • the agent that distinguishes cytosine from 5-methylcyctosine can be an agent that inhibits DNA methyltransferase activity (e.g., 5-aza-cytidine). Treatment of cells with an agent that inhibits DNA methyltransferase activity will result in loci that were methylated to lose their methylation, thus distinguishing cytosine from 5-methylcytosine.
  • the in vitro transcription step in the method of the invention converts the DNA into RNA.
  • the in vitro transcription step typically involves using primers that are complementary to sequences located in the adaptors that are ligated to the fragmented DNA.
  • the detection of the products of the in vitro transcription step in the method of the invention can be accomplished in a number of ways.
  • One particular method is hybridization of the RNA to a microarray designed to have probes corresponding to various permutations of sequences that can contain 5-methylcytosine and cytosine in the particular genome of interest.
  • the in vitro transcription step can be performed under conditions suitable for incorporating labeled nucleotides into the RNA that allow their later identification on a microarray.
  • the RNA synthesized in the in vitro transcription step is processed further depending on the type of array used, before being hybridized to the microarray.
  • Another method for detecting the products of the in vitro transcription step can involve DNA sequencing.
  • the skilled artisan is familiar with and can readily implement methods for sequencing nucleic acids, including DNA.
  • the invention therefore, allows for the determination of the methylation profile of the genome of a cell or group of cells.
  • the methylation profile of a cell, tissue or fluid can be correlated with specific phenotypic information and/or compared to "normal" methylation profiles.
  • the methylation profile can also be used for diagnostic and/or prognostic information.
  • a method for determining the methylation status of DNA.
  • the method involves obtaining or providing a sample having DNA.
  • the DNA sample is fragmented to provide DNA fragments.
  • the next step involves the treatment of the fragmented DNA with a deaminating agent (e.g., bisulphite) that can deaminate cytosines that do not have a 5-methyl group (in effect converting cytosine to uracil).
  • a deaminating agent e.g., bisulphite
  • the resulting DNA is converted to double-stranded DNA by a primer extension reaction.
  • the double-stranded DNA is then subjected to in vitro transcription conditions to yield RNA.
  • the RNA is then detected to determine its identity and therefore provide information as to whether a particular position has a cytosine or 5-methylcytosine.
  • the RNA is processed and hybridized to an array to determine the methylation pattern of the DNA.
  • the method of the invention involves determining DNA methylation status by (1) obtaining a sample having genomic DNA, (2) fragmenting the genomic DNA with two or more cytosine methylation insensitive restriction enzymes having different recognition sites, (3) ligating adaptors having 5 '-methyl protected cytosine to the fragmented DNA, (4) treating the ligated DNA fragments with a deaminating agent, (5) synthesizing double-stranded DNA from the single-stranded DNA obtained from the previous step, (6) performing in vitro transcription on double-stranded DNA formed from the ligated deaminated DNA fragments to produce RNA, and (6) hybridizing the RNA to a microarray to determine the identity of the sequences obtained from the in vitro transcription, thereby determining the DNA methylation status of the DNA.
  • kits useful for genome-wide screening of methylation status The kits can also be used for diagnostic and prognostic purposes.
  • FIG. 1 is a diagram outlining one specific implementation of the method of the invention.
  • Step A is the provision of genomic DNA.
  • Step B involves the fragmentation of the genomic DNA with two restriction enzymes that are not methylation sensitive.
  • Step C is the ligation of two different adaptors, each adaptor containing a different promoter sequence (one for T7 and the other for T3), and each adaptor engineered to be specific for one of the overhang sequence created from the fragmentation. If the adaptors contain cytosine then they are in the form of 5-methylcytosines.
  • Step D involves the treatment of the fragmented DNA that has been ligated to the adaptors with bisulphite to convert cytosines to uracils, whereas 5-methylcytosines remain 5-methylcytosines.
  • Step D the DNA is no longer able to hybridize to it complementary strand because the strands are no longer complementary after conversion of cytosines to uracils.
  • Step E involves converting the single-stranded DNA from step D into double-stranded DNA with a primer extension reaction, using promoters engineered into the ligated adaptors.
  • Step F involves the transcription of the DNA from step E into RNA, using specific transcriptase binding sites engineered into the ligated adaptor, and incorporation of label.
  • Step G involves hybridization of the RNA to an array having a plurality of probes useful distinguishing cytosine and 5-methylcytosine in the original DNA.
  • FIG. 2 illustrates one specific implementation of the method of the invention relating to steps following the ligation of the adaptors to the fragmented DNA.
  • the "C"s marked with an asterisk (*) represent 5-methylcytosine.
  • Step A represents the bisulphite treatment of DNA with a Forward ORI sequence and a reverse ORI sequence to exemplify the method for one particular sequence. The bisulphite treatment converts the cytosines (C) to uracil (U) whereas the 5-methylcytosines remain 5-methylcytosines and the strands are no longer complementary.
  • Step B using primers based on the promoter sequences ligated into the adaptors, double-stranded DNA is synthesized using a primer extension reaction.
  • Step C involves synthesis of RNA from the double-stranded DNA using an in vitro transcription reaction (with specific transcriptase binding sites engineered into the ligated adaptor) with incorporation of label into the RNA that is synthesized.
  • the final product is complementary to the forward ORI sequence after bisulphite treatment. As shown in FIG 3, if the target sequence has its cytosine methylated it will hybridize to one probe corresponding to the methylated state (+) whereas it will not hybridize to the probe corresponding to the non-methylated state (-).
  • FIG. 3 illustrates a specific example of how probes may be designed for use in the methods of the invention.
  • Probe design involves starting with a sequence and determining in silico the effects of a theoretical bisulphite treatment.
  • A corresponds to the forward ORI sequence with complete cytosine methylation and modification after bisulphite treatment
  • B corresponds to the forward ORI sequence with no cytosine methylation and modification after bisulphite treatment
  • C corresponds to the reverse ORI sequence with complete cytosine methylation and modification after bisulphite treatment
  • (D) corresponds to the reverse ORI sequence with no cytosine methylation and modification after bisulphite treatment.
  • FIG. 1 corresponds to the forward ORI sequence with complete cytosine methylation and modification after bisulphite treatment
  • B corresponds to the forward ORI sequence with no cytosine methylation and modification after bisulphite treatment
  • C corresponds to the reverse ORI sequence with complete cytosine methylation and modification after bisulphite
  • FIG. 4 shows the results of the experiments described in Example 1 with a synthetic ER-alpha (FIG. 4A) promoter with the y-axis having the Cy5 signal intensity corresponding to the methylated sample and the x-axis having the Cy3 signal intensity corresponding to the non-methylated signal intensity (the light gray triangles are for the probes to the non-methylated sequences whereas the darker diamonds are for the probes to the methylated sequence); the pl6INK4a promoter (FIG.
  • FIG. 5 shows the results of the experiment carried out to test the applicability of the method for genome-wide determination of methylation patterns as described in Example 2.
  • the experiments were designed to examine the promoters of six genes: pl6INK4a, ER-alpha, E-cadherin, MGMT, GSTPl, and APC from genomic DNA samples with the y-axis having the Cy5 signal intensity corresponding to the methylated sample and the x-axis having the Cy3 signal intensity corresponding to the non-methylated signal intensity (the light gray triangles are for the probes to the non-methylated sequences whereas the darker circles are for the probes to the methylated sequence).
  • the results show that the methylation status of a number of promoters can be distinguished use complex biological samples using the methods of the invention.
  • MODE(S) FOR CARRYING OUT THE INVENTION The invention is based on the development of a method useful for detecting methylation patterns. Methylation patterns are sometimes referred to as the methylome, methylation fingerprint, methylation status, or methylation profile.
  • the invention relates to the discovery of methods and compositions useful for detecting and characterizing the methylation of nucleic acids, e.g., DNA.
  • the method can be used for assessing the DNA methylation of genomic DNA (including DNA derived from genomic DNA) and DNA from other sources like circulating cell-free DNA.
  • the method is useful in numerous applications including the diagnosis and prognosis of diseases having altered DNA methylation patterns. Other applications include the correlation of the methylation of specific biomarkers with disease, increased susceptibility to disease, and prognosis.
  • the method of the invention is useful for biomarker discovery.
  • the invention can be used to identify specific biomarkers associated with phenotypes and for establishing methylation profiles (or methylation status).
  • the method of the invention can also be used for detecting the methylation profiles of tissues obtained from biopsy or surgery.
  • the method can also involve detection of methylated CpG islands in easily accessible biological materials such as serum and other fluids.
  • the method of the invention is also useful for the early diagnosis of disease and cancer.
  • the method and compositions of the invention are therefore generally useful for determining genome- wide methylation patterns.
  • the method of the invention involves determining the methylation of a nucleic acid by treating the nucleic acid with an agent capable of distinguishing 5-methylcytosine and cytosine, subjecting the resulting nucleic acid to in vitro transcription, and detecting the products of the in vitro transcription to determine the methylation of the nucleic acid.
  • the method of the invention involves (1) providing a sample having nucleic acid, (2) treating the nucleic acid sample with an agent capable of distinguishing a base and its methylated version, (3) subjecting the resulting nucleic acid to conditions sufficient for reverse transcription and (4) detecting the products of the reverse transcription to determine the methylation of the nucleic acid.
  • the nucleic sample is fragmented prior to treatment with the agent that a base and its methylated version.
  • adaptors are ligated to the fragment nucleic acid prior to treatment with the agent that distinguishes a base and its methylated version.
  • the detecting step comprises processing the products of the in vitro transcription step and hybridizing them to a microarray.
  • the base is cytosine and the methylated version is 5-methylcytosine.
  • the method of the invention involves (1) providing a sample of DNA, (2) treating the DNA sample with an agent capable of distinguishing 5-methylcytosine and cytosine, thereby creating single-stranded DNA, (3) subjecting the resulting single-stranded DNA to conditions sufficient to create double-stranded DNA, (4) subjecting the double-stranded DNA to conditions sufficient for in vitro transcription, and (5) detecting the products of the in vitro transcription to determine the methylation of the DNA.
  • the DNA sample is fragmented prior to treatment with the agent that distinguishes cytosine and 5 '-methylcytosines.
  • step 3 is accomplished by a primer extension reaction using primers complementary to adaptors that are ligated to the fragmented DNA.
  • the detecting step comprises processing the products of the in vitro transcription step and hybridizing them to a microarray.
  • the in vitro transcription step involves linear amplification of DNA to produce RNA.
  • the lack of DNA amplification step in this aspect ensures fidelity and avoids the loss of methylation status information caused by amplification.
  • the sample of nucleic acid used in the method of the invention can be obtained from any cell (or cells), a tissue, a fluid, or composition having methylated nucleic acid.
  • the nucleic acid sample is genomic DNA obtained from a cell or cells suspected of being cancerous.
  • the cells are derived from the culture of a cell line, hi some aspects of the invention, the tissue is derived from a xenograft.
  • the genomic DNA is obtained from a body fluid like serum, plasma, saliva, urine, or other bodily fluids.
  • the DNA is obtained from a biopsy
  • the sample is from a body fluid chosen from blood serum, blood plasma, fine needle aspirate of the breast, biopsy of the breast, ductal fluid, ductal lavage, feces, urine, sputum, saliva, semen, lavages, biopsy of the lung, bronchial lavage or bronchial brushings.
  • the sample is from a tumor or polyp.
  • the sample is a biopsy from lung, kidney, liver, ovarian, head, stomach, neck, thyroid, bladder, cervical, colon, endometrial, esophageal, prostate or skin tissue.
  • the sample is from cell scrapes, washings, or resected tissues.
  • the methylation status of at least one cytosine, CpG island, or promoter is compared to the methylation status of a control locus.
  • the control locus is an endogenous control (e.g., comparison of tumor tissue to healthy tissue of the same origin as the tumor).
  • the control locus is an exogenous control (e.g., comparison of DNA from tissue of one individual to the DNA from the same tissue from a different individual).
  • the methylation status of normal tissue is compared to the methylation status of disease tissue.
  • comparisons can be employed with the method of the invention, including comparing normal tissue from a group of subjects to matched disease tissue from a group of patients.
  • the methylation status of prostate cancer tissue obtained from patients having prostate cancer can be compared to normal non-cancerous prostate tissue (either derived from the sample population of patients and/or from healthy patients).
  • Another example can use other tissue besides the diseased tissue: skin macrophages from healthy patients compared to skin macrophages from patients having disease (e.g., lung cancer).
  • changes in the methylation status between the normal and diseased groups can identify biomarkers correlated with the characteristic of interest (e.g., diagnosis, prognosis, likelihood of response to a therapeutic, etc.).
  • the invention therefore allows for the determination of the methylation profile of the genome of a cell or group of cells.
  • the methylation profile of a cell, tissue or fluid can be correlated with specific phenotypic information and/or compared to "normal" methylation profiles to identified patterns or specific markers associated with particular phenotypic information.
  • One important step in the method of the invention involves contacting the DNA sample with an agent capable of distinguishing cytosine from 5-methylcytosine.
  • the agent that distinguishes cytosine and 5-methylcytosine changes the base pairing characteristic of one of these bases. Any number of treatments can accomplish this, including bisulphite treatment which converts cytosine to uracil (or a base with similar base-pairing properties as uracil) and leaves 5-methylcytosine unchanged. Any number of treatments can accomplish this, including bisulphite treatment which converts cytosine to uracil and leaves 5-methylcytosine unchanged.
  • Bisulphite treatment can involve treatment of the DNA with a bisulphite solution under alkaline conditions for a time sufficient to deaminate cytosine but not 5-methylcytosine.
  • Descriptions of bisulfite treatment methods can be found in, e.g., U.S. Patents 6,265,171 and 6,331,393; Boyd and Zon, Anal. Biochem. 326:278-280, 2004.
  • the in vitro transcription step in the method of the invention converts the DNA into RNA.
  • In vitro transcription normally requires double-stranded DNA as a template.
  • extension of single-stranded DNA obtained from the treatment of the DNA with from the previous step can be accomplished using primers complementary to sequence in the adaptors that were previously ligated to the fragmented DNA.
  • the double strand DNA is template for RNA polymerase, e.g., transcriptase, which binds to a specific promoter sequence also included in the adaptor sequence.
  • Other components of the in vitro transcription include the necessary nucleotides (rNTPs), and an appropriate reaction buffer. Detection of the products of the in vitro transcription can be accomplished in a number of ways.
  • RNA produced from the in vitro transcription step is processed prior to hybridizing to the microarray (e.g., fragment and purified).
  • the microarray e.g., fragment and purified.
  • silico simulations of the treatment of DNA according to the method of the invention can be performed to design probes specific for distinguishing whether or not a particular position in the DNA is methylated or not.
  • Procedures for hybridizing and detecting sequences on a microarray are known to the skilled artisan and depend on the microarray platform used. Such procedures, for example, can involve dual hybridization and/or co-hybridization protocols.
  • the microarrays for use in the invention can be one-dimensional, two-dimensional and/or a three-dimensional arrangement of addressable regions bearing a particular chemical moiety or moieties (such as ligands, e.g., biopolymers such as polynucleotide or oligonucleotide sequences (nucleic acids) associated with that region.
  • ligands e.g., biopolymers such as polynucleotide or oligonucleotide sequences (nucleic acids) associated with that region.
  • the arrays used in the embodiments are arrays of polymeric binding agents, where the polymeric binding agents may be any one or more of: polypeptides, proteins, nucleic acids, polysaccharides, synthetic mimetics of such biopolymeric binding agents, etc.
  • the arrays are arrays of nucleic acids, examples of which include, but are not limited to, oligonucleotides, polynucleotides, cDNAs, mRNAs, synthetic mimetics thereof, and the like.
  • the nucleic acids may be covalently attached to the arrays at any point along the nucleic acid chain, but are generally attached at one of their termini (e.g., the 3 ' or 5' terminus). Methods for manufacturing and using arrays are known to the skilled artisan and are commercially available.
  • methylation insensitive enzyme refers to any enzyme that will cut a nucleic acid sequence at a CpG site with or without a 5-methylcytosine. In other words, a methylation insensitive enzyme will cleave a methylation restriction site independent of its methylation status.
  • methylation insensitive enzyme examples include Mspl, Taql, Xmal, and FspBI.
  • methylation generally refers to cytosine methylation at position C5 of cytosine (5-methylcytosine).
  • methylation generally refers to cytosine methylation at position C5 of cytosine (5-methylcytosine).
  • methylation profile refers to a set of data representing the methylation state of one or more loci within a molecule of DNA from e.g., the genome of an individual or cells or tissues from an individual (including for example cell free DNA).
  • the profile can indicate the methylation state of every chosen base in an individual, can have information regarding a subset of the bases (e.g., the methylation state of specific promoters or quantity of promoters or specific sequences within the promoter) in a genome, or can have information regarding regional methylation density of each locus.
  • methylation status refers to the presence, absence and/or quantity of methylation at a nucleotide or nucleotides within a portion of DNA.
  • the methylation status of a particular DNA sequence can indicate the methylation state of every base in the sequence or can indicate the methylation state of a subset of the bases (e.g., whether the specific bases are cytosine or 5-methylcytosine) within the sequence.
  • Methylation status can also indicate information regarding regional methylation density within the sequence without specifying the exact location.
  • ligation refers to any process of forming phosphodiester bonds between two or more polynucleotides, such as those comprising double-stranded DNAs. Techniques and protocols for ligation may be found in standard laboratory manuals and references. Sambrook et al., In: Molecular Cloning. A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989); and Maniatis et al., pg. 146.
  • probe refers to any nucleic acid or oligonucleotide that can form a hybrid structure with a sequence of interest in a target gene region (or sequence) due to complementarily of at least one sequence in the probe with a sequence in the target region.
  • nucleic acid refers to nucleic acid regions, nucleic acid segments, primers, probes, amplicons and oligomer fragments.
  • the terms are not limited by length and are generic to linear polymers of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. These terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • a nucleic acid, polynucleotide or oligonucleotide can comprise, for example, phosphodiester linkages or modified linkages including, but not limited to phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
  • phosphodiester linkages or modified linkages including, but not limited to phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothi
  • CpG Island refers to any DNA region wherein the GC composition is over 50% in a "nucleic acid windows" having a minimum length of 200 bp nucleotides and a CpG content higher than 0.6.
  • promoter refers to a sequence of nucleotides that reside on the 5' end of a gene's open reading frame. Promoters generally comprise nucleic acid sequences which bind with proteins such as, but not limited to, RNA polymerase and various histones. Some promoters are not completely to the 5 ' of a genes open reading frame: these promoters are also within the scope of the invention.
  • a method for determining the methylation status of DNA.
  • the method involves obtaining or providing a sample having DNA.
  • Any sample or source of DNA can be used in this embodiment.
  • the DNA can be derived from a fluid sample, a tissue sample, a cell culture, cells, an aspirate, a biopsy, or a tissue section.
  • the DNA can be derived from various types of eukaryotic organisms including mammals (e.g., human) and plants.
  • the DNA sample is fragmented to provide DNA fragments that can be further processed for use in a subsequent in vitro transcription reaction.
  • the fragments suitable for in vitro transcription are prepared by treating the DNA sample with one or more restriction enzymes.
  • the conditions and restriction enzyme(s) used for fragmenting the DNA are chosen to produce fragments suitable for in vitro transcription.
  • Adaptors, having promoters for in vitro transcription, are then ligated to the fragmented DNA.
  • the next step involves the treatment of the DNA with a deaminating agent (e.g., alkaline bisulphite treatment) that can deaminate cytosines that do not have a 5 '-methyl group.
  • a deaminating agent e.g., alkaline bisulphite treatment
  • the resulting single-stranded DNA is converted to double-stranded DNA by primer extension reactions.
  • the double-stranded DNA is then subjected to in vitro transcription conditions to yield RNA.
  • the RNA is then processed and hybridized to an array to determine the methylation pattern of the DNA.
  • no PCR DNA amplification step or exponential amplification step is used in the specific method of this embodiment.
  • the method of the invention involves determining DNA methylation status by (1) obtaining a sample having genomic DNA, (2) fragmenting the genomic DNA with two or more methylation insensitive restriction enzymes having different recognition sites, (3) ligating adaptors having 5 '-methyl protected cytosine to the fragmented DNA, (4) treating the ligated DNA fragments with deaminating agent, (5) performing in vitro transcription on double-stranded DNA formed from the ligated deaminated DNA fragments, and (6) hybridizing the probes to an microarray for determining the identity if the sequences obtained from the in vitro transcription, thereby determining the DNA methylation status of the DNA.
  • no PCR DNA amplification step or exponential amplification step is used in the specific method of this embodiment.
  • the invention provides a genome-wide screening method for determining the methylation status of genomic DNA.
  • the method involves: (a) providing genomic DNA (gDNA),
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the method is as follows. First genomic DNA (gDNA) is provided.
  • the gDNA can be extracted from a sample using any suitable method.
  • the gDNA is incubated with one or more restriction enzymes to give digested genomic DNA.
  • the ends of the digested gDNA are ligated with phosphorylated and cytosine-methylated adaptors to give digested gDNA with adaptors.
  • the digested gDNA ligated with adaptors is then treated with bisulphite under conditions sufficient to deaminate cytosine bases that are not methylated. This steps yields single-stranded DNA (sDNA).
  • sDNA single-stranded DNA
  • the complementary strand of the sDNA is then synthesized to give double-stranded DNA (dsDNA) which can be the substrate for a subsequent in vitro transcription reaction.
  • dsDNA double-stranded DNA
  • the complementary strand can be synthesized using a primer extension reaction with primers based on the sequences contained in the adaptor.
  • the template double strand DNA is in vitro transcribed to synthesize RNA.
  • the RNA is then hybridized to an array to determine the methylation status of the gDNA.
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the invention provides a genome-wide screening method for determining methylation status of genomic DNA.
  • the method involves providing genomic DNA (gDNA).
  • the gDNA can be extracted from a sample using any suitable method.
  • the gDNA is incubated with one or more restriction enzymes to give digested genomic DNA.
  • the ends of the digested gDNA are ligated with phosphorylated and cytosine-methylated adaptors suitable to give digested gDNA with ligated adaptors.
  • the digested gDNA ligated with adaptors is treated with bisulphite under conditions sufficient to deaminate cytosines that are not methylated. This steps yields single-stranded DNA (sDNA).
  • the complementary strand of the bisulphite treated sDNA is then synthesized to give template double-stranded DNA (dsDNA).
  • dsDNA template double-stranded DNA
  • the complementary strand can be synthesized using a primer extension reaction with primers based on the sequence contained in the adaptor.
  • the template double strand DNA is transcribed to RNA.
  • the resulting RNA is then hybridized to an array to determine the methylation status of the gDNA.
  • no PCR DNA amplification step or exponential amplification step is used in the specific method of this embodiment.
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the method is as follows. Genomic DNA is incubated with two endonucleases having restriction sites that yield overhangs with different sequences.
  • the endonuclease treatment can be at the same time in the same reaction or the treatment can be sequential.
  • the endonucleases can be tetracutters (e.g., Taql and Bfal).
  • the fragmented genomic DNA is then contacted with adaptors under conditions sufficient to ligate the adaptors to the fragmented DNA. Two types of adaptors are used in this step, and if either contains cytosine these cytosines are in the form of 5-methylcytosine to protect from deamination in subsequent bisulphite treatment.
  • the two types of adaptors are designed so that one type can be specifically ligated to a site corresponding to one endonuclease site and the other type can be specifically ligated to the other endonuclease site.
  • the adaptor ligated fragmented genomic DNA is then treated with bisulphite to deaminate cytosine bases but not 5-methyl cytosines. This step yields bisulphite treated single-stranded DNA (sDNA).
  • sDNA single-stranded DNA
  • the complementary strand of the bisulphite treated sDNA is synthesized using a primer extension reaction based on sequences contained in the adaptors to give template dsDNA.
  • the template dsDNA is then transcribed into RNA using promoters sequences engineered into the adaptors and conditions sufficient for transcription.
  • RNA is then hybridized to an array to determine the methylation status of the genomic DNA.
  • no PCR DNA amplification step or exponential amplification step is used in the specific method of the invention prior to treatment of the DNA with bisulphite.
  • no PCR DNA amplification step or exponential amplification step is used in the specific method of this embodiment.
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the present invention provides methods for diagnosing or predicting a cancer by genome-wide methylation profiling.
  • the method of this embodiment can comprise (1) obtaining a test sample from cells or tissue, (2) obtaining a control sample from cells or tissue that is normal, and (3) detecting or measuring in both the test sample and the control sample the genome-wide methylation profile using the method of the invention. If the methylation profile of test sample is altered compared to the control sample (or value), this indicates a cancer or a precancerous condition in the test sample cells. If the level methylation of one or more tumor suppressors is higher in the test sample as compared to the control sample (or value), this indicates a cancer or a precancerous condition in the test sample cells or tissue.
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the method of the invention is used to determine whether two or more tumors are more likely to have arisen independently or more likely to be clonal (e.g., primary and metastasis). According to this method the methylation profiles determined by the method of the invention are compared. Methylation profiles that are substantially similar indicate that the tumors are more likely to be clonal whereas methylation profiles that are substantially different are more likely to have originated independently. In one embodiment, the method of the invention comprises determining the methylation status of one or more miRNA cytosines, CpG dinucleotides, CpG islands, and/or promoters.
  • the method of this embodiment involves obtaining or providing a sample having nucleic acid (e.g., genomic DNA) suspected of having miRNA (e.g., nucleic acid involved in the regulation of expression of the miRNA).
  • the DNA sample is fragmented to provide DNA fragments that can be further processed for use in subsequent in vitro transcription reaction(s).
  • the fragments suitable for in vitro transcription are prepared by treating the DNA sample with one or more restriction enzymes. The conditions and restriction enzyme(s) used for fragmenting the DNA are chosen to produce fragments suitable for in vitro transcription. Adaptors, having promoters for in vitro transcription, are then ligated to the fragmented DNA.
  • the next step involves the treatment of the DNA with a deaminating agent (e.g., alkaline bisulphite treatment) that can deaminate cytosines that do not have a 5 '-methyl group.
  • a deaminating agent e.g., alkaline bisulphite treatment
  • the resulting single-stranded DNA is converted to double-stranded DNA by primer extension reactions.
  • the double-stranded DNA is then subjected to in vitro transcription conditions to yield RNA.
  • the RNA is then processed and hybridized to an array to determine the methylation pattern of the DNA of one or more miRNA promoters. Examples of miRNA genes which have altered methylation status and/or profiles in cancer are known in the art.
  • miRNA are epigenetically regulated in Acute Lymphoblastic Leukemia where altered expression levels associated with altered CpG methylation of the promoters of these miRNA (Hsa-miR-9, Hsa-miR-lOb, Hsa-miR-34, Hsa-miR-124, Hsa-miR-132, Hsa-miR-196b, Hsa-miR-203, and Hsa-miR-212); Lujambo et al.
  • the methylation status and/or profile of cytosines in any nucleic acid sequence associated with a miRNA sequence can be determined using the method of the invention.
  • probes for different methylation states of miRNA can be created and used to detect methylation of miRNA promoter sequences, cytosines, CpG dinuccleotides, and/or CpG islands involved in regulation of expression of the miRNA.
  • the methylation status of one or more miRNA chosen from Hsa-miR-9, Hsa-miR-10b, Hsa-miR-34, miR-34a, Hsa-miR-124, Hsa-miR-132, Hsa-miR-196b, Hsa-miR-203, Hsa-miR-212, miR-148a, miR-34b/c, miR-9, and miR-1-1 is determined using the methods of the invention, hi specific aspects of the method of this embodiment, the DNA is not exponentially amplified.
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the method is used to measure the methylation status of one or more markers in fetal DNA.
  • the fetal DNA is obtained from maternal plasma.
  • the fetal DNA is analyzed for prenatal diagnosis, hi one aspect of this embodiment, the methylation status or profile of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more cytosines, promoters, and/or CpG islands are determined according to the methods of the invention. In one aspect of this embodiment, the methylation status or profile of from 2 to 1000, 3 to 1000, 4 to 1000, 5 to 1000, 6 to 1000, 7 to 1000, 8 to 1000, 9 to 1000, or 10 to 1000 cytosines, promoters, and/or CpG islands are determined according to the methods of the invention. Chim et al.
  • the method comprises detecting the presence or absence of fetal trisomy 21 in DNA obtained from maternal plasma.
  • the method comprises analyzing the methylation profile one or more promoters, CpG islands, and/or cytosines that are differentially methylated in maternal as compared to fetal DNA.
  • the one or more promoters, CpG islands, and/or cytosines that are differentially methylated in maternal as compared to fetal DNA are on chromosome 21.
  • the one or more promoters, CpG islands, and/or cytosines that are differentially methylated in maternal as compared to fetal DNA are on chromosome 21 are chosen from CGI009, CGI023, CGI027, CGI028, CGI045, CGI051, CGI052, CGI071, CGI105, CGI109, CGI113, CGI127, CGI149, CGI40, CGI43, CGI084, CGI092, CGI093, CGI136, CGI137, CGI139, and CGIl 40.
  • determining the methylation of the DNA further comprises comparing the sequence of DNA treated with an agent capable of distinguishing 5-methylcyctosine from cytosine to DNA not treated with an agent capable of distinguishing 5-methylcytosine from cytosine.
  • the method is used to measure the methylation status of one or more markers for diagnosing prostate cancer.
  • the sample to be analyzed is obtained from circulating DNA obtained from blood of an individual.
  • the sample to be analyzed is obtained from urine or urine sediment, hi yet another aspect, the sample to be analyzed is obtained from a patient suspected of having or desiring screening for prostate cancer (e.g., biopsy).
  • the sample having DNA that is to be analyzed with an agent capable of distinguishing 5-methylcytosine and cytosine e.g., bisulphite treatment
  • subjecting the resulting DNA to in vitro transcription and (4) detecting the products of the in vitro transcription to determine the methylation of the DNA.
  • the one or more markers that are analyzed are chosen from RASSFl, RARB2, and GSTPl. In another specific aspect, the markers that are analyzed are chosen from pi 6, ARF, MGMT, and GSTPl.
  • the primers used for in vitro transcription are based on sequences contained in adaptor sequences that are ligated to the DNA in the sample, hi one aspect of this embodiment, the genomic DNA is not amplified by PCR. Hoque et al. ((2005) J. Clin. Oncol. 23:6569-75) and Sunami et al. ((2009) Clin. Chem. 55:3) describe markers for detecting prostate cancer.
  • the method of this embodiment is used to measure the methylation status of one or more markers for prognosis and/or risk stratification of prostate cancer.
  • the method of the invention is used to determine the methylation status of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 tumor suppressor promoters.
  • the method of the invention is used to determine the methylation status of from 1 to 1000 promoters, 2 to 1000 promoters, 3 to 1000 promoters, 4 to 1000 promoters, 5 to 1000 promoters, 6 to 1000 promoters, 7 to 1000 promoters, 8 to 1000 promoters, 9 to 1000 promoters, or 10 to 1000 promoters.
  • the one or more tumor suppressors are chosen from p53; the retinoblastoma gene, commonly referred to as RbI; the adenomatous polyposis of the colon gene (APC); familial breast/ovarian cancer gene I (BRCAl); familial breast/ovarian cancer gene 2 (BRC A2); CDHl cadherin 1 (epithelial cadherin or E-cadherin) gene; cyclin-dependent kinase inhibitor 1C gene (CDKNlC, also known as p57, KIP2 or BWS); cyclin-dependent kinase inhibitor 2 A gene (CDKN2A also known as pi 6 MTSl (multiple tumor suppressor 1), TP 16 or INK4); familial cylindromatosis gene (CYLD; formerly known as EAC (epithelioma adenoides cysticum)); ElA-binding protein gene (p300); multiple exostosis type 1 gene (EXX)
  • EAC
  • HNPCC human non-polyposis colorectal cancer
  • HNPCC2 formerly referred to as COCA2 (colorectal cancer 2) and FCC2
  • MSH2 also called HNPCC (hereditary non-polyposis colorectal cancer) or HNPCCl and formerly known as COCAl (colorectal cancer 1) and FCCl
  • NFl neurofibromatosis type 1 gene
  • NF2 neurofibromatosis type 2 gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRKARl or TS ⁇ 1 tissue-specific extinguisher I
  • the method of the invention is used to determine the methylation status and/or profile of the promoters of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 oncogenes.
  • the method of the invention is used to determine the methylation status of from 1 to 1000 oncogene promoters, 2 to 1000 oncogene promoters, 3 to 1000 oncogene promoters, 4 to 1000 oncogene promoters, 5 to 1000 oncogene promoters, 6 to 1000 oncogene promoters, 7 to 1000 oncogene promoters, 8 to 1000 oncogene promoters, 9 to 1000 oncogene promoters, or 10 to 1000 oncogene promoters.
  • the one or more oncogenes are chosen from K-RAS, H-RAS, N-RAS, EGFR, MDM2, RhoC, AKTl, AKT2, MEK (also called MAPKK), c-myc , n-myc, beta-catenin, PDGF, C-MET, PIK3CA, CDK4, cyclin Bl, cyclin Dl, estrogen receptor gene, progesterone receptor gene, ErbBl, ErbB2 (also called HER2), ErbB3, ErbB4, TGF-alpha, TGF-beta, ras-GAP, She, Nek, Src, Yes, Fyn, Wnt, BCL 2 , and Bmil.
  • the method comprises determining the methylation status and/or profile of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100, 150, 200, 250, 300, 400, 500, 750, or 1000 cytosines in a DNA sample.
  • the method comprise determining the methylation status of from 1 to 10,000 cytosines, 2 to 10,000 cytosines, 3 to 10,000 cytosines, 4 to 10,000 cytosines, 5 to 10,000 cytosines, 6 to 10,000 cytosines, 7 to 10,000 cytosines, 8 to 10,000 cytosines, 9 to 10,000 cytosines, or 10 to 10,000 cytosines.
  • the method comprises determining the methylation status and/or profile of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100, 150, 200, 250, 300, 400, 500, 750, or 1000 promoters in a DNA sample.
  • the method of the invention is used to determine the methylation status of from 1 to 1000 promoters, 2 to 1000 promoters, 3 to 1000 promoters, 4 to 1000 promoters, 5 to 1000 promoters, 6 to 1000 promoters, 7 to 1000 promoters, 8 to 1000 promoters, 9 to 1000 promoters, or 10 to 1000 promoters.
  • the method comprises determining the methylation status and/or profile of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100, 150, 200, 250, 300, 400, 500, 750, or 1000 CpG islands within a DNA sample.
  • the method of the invention is used to determine the methylation status of from 1 to 1000 CpG islands, 2 to 1000 CpG islands, 3 to 1000 CpG islands, 4 to 1000 CpG islands, 5 to 1000 CpG islands, 6 to 1000 CpG islands, 7 to 1000 CpG islands, 8 to 1000 CpG islands, 9 to 1000 CpG islands, or 10 to 1000 CpG islands.
  • the invention provides a microarray for determining the methylation status and/or profile of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 tumor suppressor promoters.
  • the invention provides a microarray for determining the methylation status of from 2 to 1000 tumor suppressor promoters, 3 to 1000 tumor suppressor promoters, 4 to 1000 tumor suppressor promoters, 5 to 1000 tumor suppressor promoters, 6 to 1000 tumor suppressor promoters, 7 to 1000 tumor suppressor promoters, 8 to 1000 tumor suppressor promoters, 9 to 1000 tumor suppressor promoters, or 10 to 1000 tumor suppressor promoters.
  • the microarray is designed to have probes for determining the methylation status (or profile) of each promoter for each tumor suppressor, according to the method of the invention.
  • one or more of the tumor suppressors are chosen from p53; the retinoblastoma gene, commonly referred to as RbI; the adenomatous polyposis of the colon gene (APC); familial breast/ovarian cancer gene I (BRCAl); familial breast/ovarian cancer gene 2 (BRCA2); CDHl cadherin 1 (epithelial cadherin or E-cadherin) gene; cyclin-dependent kinase inhibitor 1C gene (CDKNlC, also known as p57, KIP2 or BWS); cyclin-dependent kinase inhibitor 2 A gene (CDKN2A also known as pl6 MTSl (multiple tumor suppressor 1), TP 16 or INK4); familial cylindromatosis gene (CYLD;
  • HNPCC human non-polyposis colorectal cancer
  • HNPCC2 formerly referred to as COC A2 (colorectal cancer 2) and FCC2
  • MSH2 also called HNPCC (hereditary non-polyposis colorectal cancer) or HNPCCl and formerly known as COCAl (colorectal cancer 1) and FCCl
  • NFl neurofibromatosis type 1 gene
  • NF2 neurofibromatosis type 2 gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRXARlA protein kinase A type 1, alpha, regulatory subunit gene
  • PRKARl or TSEl tissue-specific extinguisher I
  • the one or more tumor suppressors are chosen from, APC, BRCAl, BRCA2, CDHl, CDKN2A, DCC, DPC4 (SMAD4), MADR2/JV18 (SMAD2), MENl, MLHl, MSH2, MTSl, NFl, NF2, PTCH, p53, PTEN, RBl, TSCl, TSC2, VHL, WRN, and WTl.
  • the one or more tumor suppressors are chosen from CDHl (E_Cadherin), p 16INK4a, APC, GSTP 1 , and MGMT.
  • the invention provides a microarray for determining the methylation status and/or profile of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 oncogenes.
  • the microarray is designed to have probes for determining the methylation status (or profile) of each promoter for each tumor oncogene, according to the method of the invention, hi one aspect of this embodiment, the microarray has probes for detecting the methylation status or profile of from 2 to 1000 oncogene promoters, 3 to 1000 oncogene promoters, 4 to 1000 oncogene promoters, 5 to 1000 oncogene promoters, 6 to 1000 oncogene promoters, 7 to 1000 oncogene promoters, 8 to 1000 oncogene promoters, 9 to 1000 oncogene promoters, or 10 to 1000 oncogene promoters.
  • one or more of the oncogenes are chosen from K-RAS, H-RAS, N-RAS, EGFR, MDM2, RhoC, AKTl, AKT2, MEK (also called MAPKK), c-myc , n-myc, beta-catenin, PDGF, C-MET, PIK3CA, CDK4, cyclin Bl, cyclin Dl, estrogen receptor gene, progesterone receptor gene, ErbBl, ErbB2 (also called HER2), ErbB3, ErbB4, TGF-alpha, TGF-beta, ras-GAP, She, Nek, Src, Yes, Fyn, Wnt, BCL 2 , and Bmil.
  • the invention provides a method of diagnosis and/or prognosis of cancer.
  • the method comprises obtaining a sample having a nucleic acid, subjecting the nucleic acid to conditions sufficient to deaminate 5-methyl cytosine, subjecting the treated nucleic acid to intra transcription.
  • the method comprises diagnosis of prostate cancer.
  • the methylation of CpG islands in GSTPl, FLNC, RARB2, and PTX2 are determined to differentiate between prostate cancer and benign prostatic hyperplasia (Vanaja et al. (2009) Cancer Investigation DOI 10.1080/07357900802620794).
  • the method comprises PITX2, PDLIM4, KCNMAl, GSTPl, FLNC, EFS, and ECRG4 to distinguish cancers are more likely to be recurrent or less likely to be recurrent.
  • methylation of FLNC, PITX, EFS, and ECRG4 are associated with recurrent prostate cancer.
  • Methylation of individual CpG units can be used to diagnose prostate cancer e.g., RARB2_CpG_10.11, RARB2_CpG_l, RARB2_CpG_9, GSTPl_CpG_21, GSTPl_CpG_10, GSTPl_CpG_22, GSTPl_CpG_17.18, PITX2_CpG_31.32, GSTPl_CpG_19, GSTPl_CpG_8, FLNC_CpG_36.37.38, PITX2_CpG_14, PITX2_CpG_6.7, PITX2_CpG_34, GSTPl_CpG_l l, GSTPl_CpG_12.13, and PITX2_CpG_26.27.
  • RARB2_CpG_10.11, RARB2_CpG_l, RARB2_CpG_9 GSTPl_CpG
  • the method relates to the diagnosis of breast cancer.
  • the method of this aspect comprise comparing the methylation profile of nucleic acid obtained from a breast cancer patient or a patient suspected of having or desiring screening for breast cancer.
  • the methylation profile as determined by the method of the invention can be compared to the methylation profile for normal breast cells, blood cells, and/or a control value.
  • the markers analyzed for methylation are chosen from cytosines, CpG, and promoters involved in the regulation of expression of a specific gene(s).
  • the markers are chosen from GHSR, chr7-8256880, LMTK3, MGA, chrl-203610783, CD9, hATHl, STK36, h3-OST-2, FLRT2, PRDM 12, NFIX, CDX-2, CXCLl, ZBTB 8, and Hox-A7.
  • Ordway et al. (2007J PLoS ONE 2(12):el314 describe methylation markers with high sensitivity and specificity for breast cancer.
  • the invention provides a method of characterizing tumor progression (and/or diagnosing cancer) by determining hypermethylation and/or hypomethylation of DNA in a sample from a patient suspected of having cancer (or desiring screening for cancer).
  • the method comprises obtaining a cancer sample from a patient and determining the methylation status of the DNA by treating the DNA with a deaminating agent (e.g., bisulphite treatment), subjecting said treated DNA to in vitro transcription, and detecting the methylation the DNA is the sample.
  • a deaminating agent e.g., bisulphite treatment
  • DNA hypomethylation and hypermethylation have been associated with a number of cancers including lung cancer (see e.g., Anisowicz et al.
  • the invention provides a set of probes useful for the diagnosing, prognosis, and/or characterization of prostate cancer, wherein said probes are designed to detect changes in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines differentially methylated in prostate cancer according to the method of the invention.
  • the set of probes is attached to a solid support.
  • the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • the probes are designed to detect markers with differential methylation in prostate cancer
  • the method of analyzing the DNA comprises treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine (e.g., bisulphate treatment) and followed by subjecting the resulting DNA to in vitro transcription, hi a specific aspect, the DNA is not amplified prior to treatment of the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the invention provides a set of probes useful for the diagnosing, prognosis, and/or characterization of breast cancer, wherein said probes are designed to detect changes in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines differentially methylated in breast cancer according to the method of the invention.
  • the set of probes is attached to a solid support.
  • the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • the probes are designed to detect markers with differential methylation in breast cancer
  • the method of analyzing the DNA comprises treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine (e.g., bisulphate treatment) and followed by subjecting the resulting DNA to in vitro transcription, hi a specific aspect, the DNA is not amplified prior to treatment of the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the invention provides a set of probes useful for the diagnosing, prognosis, and/or characterization of colorectal cancer, wherein said probes are designed to detect changes in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines differentially methylated in colorectal cancer according to the method of the invention, hi one aspect of this embodiment, the set of probes is attached to a solid support, hi one aspect of this embodiment, the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • the probes are designed to detect markers with differential methylation in colorectal cancer
  • the method of analyzing the DNA comprises treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine (e.g., bisulphate treatment) and followed by subjecting the resulting DNA to in vitro transcription.
  • the DNA is not amplified prior to treatment of the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the invention provides a set of probes useful for the diagnosing, prognosis, and/or characterization of lung cancer, wherein said probes are designed to detect changes in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines differentially methylated in lung cancer according to the method of the invention.
  • the set of probes is attached to a solid support.
  • the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • the probes are designed to detect markers with differential methylation in lung cancer
  • the method of analyzing the DNA comprises treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine (e.g., bisulphate treatment) and followed by subjecting the resulting DNA to in vitro transcription.
  • the DNA is not amplified prior to treatment of the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the invention provides a set of probes useful for the diagnosing, prognosis, and/or characterization of ovarian cancer, wherein said probes are designed to detect changes in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines differentially methylated in ovarian cancer according to the method of the invention.
  • the set of probes is attached to a solid support.
  • the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • the probes are designed to detect markers with differential methylation in ovarian cancer
  • the method of analyzing the DNA comprises treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine (e.g., bisulphate treatment) and followed by subjecting the resulting DNA to in vitro transcription.
  • the DNA is not amplified prior to treatment of the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the invention provides a set of probes useful for prenatal diagnose and/or prognosis, wherein said probes are designed to detect in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines that are differentially methylated in fetal DNA as compared to maternal DNA according to the method of the invention.
  • the DNA that is analyzed is circulating DNA (e.g., obtained from maternal plasma).
  • the set of probes is attached to a solid support.
  • the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • the method comprises determining the DNA methylation of markers for trisomy 21 (Down syndrome).
  • the probes are designed to detect markers with differential methylation in fetal DNA as compared to Maternal DNA
  • the method of analyzing the DNA comprises treating the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine (e.g., bisulphate treatment) and followed by subjecting the resulting DNA to in vitro transcription.
  • the DNA is not amplified prior to treatment of the DNA with an agent capable of distinguishing 5-methylcytosine and cytosine.
  • the invention provides a set of probes useful for the diagnosing, prognosis, and/or characterization of circulating DNA in plasma, wherein said probes are designed to detect changes in the methylation profile or status of the DNA of promoters, CpG islands, and/or cytosines differentially methylated in circulating DNA in plasma in different disease states according to the method of the invention.
  • the set of probes is attached to a solid support.
  • the set of probes has from 10 to 100,000 distinct probes, from 10 to 50,000 distinct probes, from 10 to 10,000 probes, and from 10 to 1,000 probes.
  • different number of probes are designed covering from sequence of maximal concentration of CpGs (normally located around 1000 pb up-stream transcription site) to the CpG enriched sequences near transcription site.
  • the probes are between 21-27 nt long and selected to avoid non-specific cross-hybridization at the hybridization conditions: 60 0 C.
  • the methylation-insensitive restriction enzyme recognizes a restriction enzyme target sequence of 4, 5, or 6 base pairs.
  • labeling includes incorporation of nucleotide analogs containing directly detectable labeling substances, such as fluorophores, nucleotide analogs incorporating labeling substances detectable in a subsequent reaction, such as biotin or haptens, or any other type of nucleic acid labeling.
  • the nucleotide analog is selected from among the group comprising Cy3-UTP, Cy5-UTP, fluorescein-UTP, biotin-UTP, and aminoallyl-UTP.
  • RNA polymerase a sequence of nucleotides that can be recognized by an RNA polymerase and from which transcription can be initiated.
  • each RNA polymerase recognizes a specific sequence, so that the functional promoter sequence included in the adapters is chosen according to the
  • RNA polymerase used.
  • examples of RNA polymerases that can be used in the method of the present invention include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.
  • Determination of the methylation state of the sample can be performed using any nucleic acid analysis technique.
  • determination of the methylation state of the sample is carried out by hybridization of the RNA fragments obtained with the immobilized oligonucleotides on a DNA microarray, detection of the labeling incorporated in the fragments to be analyzed, and quantitative comparison of the signal values of the hybridized fragments with the values of the reference signals.
  • the methylation status of the sample is determined by analyzing and/or comparing the nucleic sequence of the bisulphite treated DNA and DNA not treated with bisulphite.
  • the invention provides a kit having (1) a component for fragmenting DNA, (2) a component for distinguishing 5-methylcytosine and cytosine, (3) an in vitro transcription component, and (4) instructions for using the components of the kit.
  • the invention provides a kit having (l) two or more methylation insensitive restriction enzymes that have different restriction sites, (2) a deaminating agent, (3) adaptors, (4) an in vitro transcription component, and (5) instructions for using the components of the kit.
  • the invention provides a kit having (l) two or more methylation insensitive restriction enzymes that have different restriction sites,
  • the invention provides a kit having (1) a component for extracting DNA, (2) two or more restriction endonucleases that are methylation insensitive and produce different overhanging sequences, (3) protected adaptors engineered to have promoters for in vitro transcription and that will hybridize to the overhangs created by the two or more restriction endonucleases, (4) a component for ligating the adaptors to the sites created by the restriction endonucleases, (5) a component for deaminating cytosines but not 5-methylcytosines, (6) an in vitro transcription component, and (7) instructions for using the components of the kit.
  • the invention provides a kit having (1) a component for extracting DNA, (2) two or more restriction endonucleases that are methylation insensitive and produce different overhanging sequences, (3) protected adaptors engineered to have promoters for in vitro transcription and that will hybridize to the overhangs created by the two or more restriction endonucleases, (4) a component for ligating the adaptors to the sites created by the restriction endonucleases, (5) a component for deaminating cytosines but not 5-methylcytosines, (6) a component capable of synthesizing dsDNA from sDNA, (7) an in vitro transcription component, and (8) instructions for using the components of the kit.
  • the invention provides a kit having (1) a component for extracting DNA, (2) a fragmenting component having two or more restriction endonucleases that are methylation insensitive and produce different overhanging sequences and reagents suitable for producing fragment DNA, (3) an adaptor component having protected adaptors engineered to have promoters for in vitro transcription and that will hybridize to the overhangs created by the two or more restriction endonucleases, (4) a component for ligating the adaptors to the sites created by the restriction endonucleases, (5) a component for deaminating cytosines but not methylcytosines, (6) a component capable of synthesizing dsDNA from sDNA, (7) an in vitro transcription component, (8) a component for preparing the RNA for hybridization to a microarray, and (9) instructions for using the components of the kit.
  • the invention provides a kit having (1) a component for extracting DNA, (2) a fragmenting component having two or more restriction endonucleases that are not methylation sensitive and produce different overhanging sequences and reagents suitable for producing fragment DNA, (3) an adaptor component having protected adaptors engineered to have promoters for in vitro transcription and that will hybridize to the overhangs created by the two or more restriction endonucleases, (4) a component for ligating the adaptors to the sites created by the restriction endonucleases, (5) a component for deaminating cytosines but not 5-methylcytosines, (6) a component capable of synthesizing dsDNA from sDNA, (7) an in vitro transcription component, (8) a component for preparing the RNA for hybridization to a microarray, (9) a microarray, and (10) instructions for using the components of the kit.
  • a component for extracting DNA refers one or more reagents useful for isolating DNA from a sample.
  • the component for extracting DNA comprises an agent for chelating divalent cations (e.g., EDTA) which help prevent degradation of the DNA, an agent for rupturing cells and remove membrane lipids (e.g., sonication and addition of a detergent), an agent for removing cellular and histone proteins bound to the DNA (e.g., a protease, or by precipitation with sodium or ammonium acetate, or by using a phenol-chloroform extraction step), an agent for precipitating DNA (cold ethanol or isopropanol), and an agent for solubilizing the DNA.
  • an agent for chelating divalent cations e.g., EDTA
  • an agent for rupturing cells and remove membrane lipids e.g., sonication and addition of a detergent
  • an agent for removing cellular and histone proteins bound to the DNA e
  • a component for fragmenting DNA refers to an agent that can fragment the DNA and yield a size range of fragments that are suitable for use in the other steps of the method.
  • Various methods and agents for fragmenting DNA are known to the skilled artisan.
  • the component for fragmenting DNA yields DNA fragment with cohesive ends.
  • the component for fragmenting DNA comprises a restriction endonuclease.
  • the component for fragmenting DNA comprises a restriction enzyme (or enzymes), that are not sensitive to DNA methylation.
  • the component for fragmenting DNA comprises two restriction enzymes.
  • the restriction enzymes are chosen from Taql and FspBI.
  • An adaptor component refers to adaptors that are designed to hybridize to cohesive ends of fragmented DNA.
  • Various adaptor and adaptor designs are known to the skilled artisan and can be used as the adaptor component.
  • all of the cytosines in the promoter are 5 '-methylated (i.e., 5-methylcytosine).
  • the adaptor component has a promoter useful for in vitro transcription, hi one specific aspect, the promoter engineered into the adaptor is a T7 or T3 promoter.
  • a component for distinguishing 5-methylcytosine and cytosine refers to an agent that modifies either 5-methylcytosine or cytosine in a way that can be detected in subsequent steps.
  • One example of a component that distinguishes 5-methylcytosine and cytosine is bisulphite treatment, which under appropriate conditions converts cytosine to uracil whereas 5-methylcytosine remains 5-methylcytosine.
  • An in vitro transcription component refers to reagents for transcribing DNA into RNA.
  • the component comprises an RNA polymerase
  • the in vitro transcription component comprises a polymerase capable of transcribing DNA into RNA and rNTPs (e.g., the 5 ribonucleotides needed for transcription
  • hi one specific aspect in vitro transcription component comprises T7 RNA Polymerase, rNTPs, and labeled CTPs.
  • Other RNA polymerases commonly used for in vitro transcription include T3 and S6.
  • a component for ligating the adaptors refers to agents that will ligate the adaptor component to the fragment DNA.
  • the component for ligating the adaptors comprises a ligase.
  • the ligase is T4 ligase.
  • a component capable of synthesizing dsDNA from sDNA refers to an agent that will synthesize double-stranded DNA from a single-stranded template.
  • the component comprises a DNA polymerase.
  • the component comprises primers specific for sequence in the adaptors.
  • the primers will hybridize to a T7 promoter, or complement thereof.
  • a component for preparing RNA for hybridization to a microarray refers to buffers and reagents that prepare the RNA for hybridization to a microarray.
  • the validation of a method for genome-wide methylation detection was accomplished by specifically determining the methylation state of three synthetic promoter fragments that have high content CpG island promoters. Specifically, fragments of the ERa, Ecadherin and pl6INK4a were used to validate the method.
  • Microarray chips were fabricated using standard techniques for microarray synthesis design. The chips were designed to include specific probes to interrogate each one of the CpG islands for three genes promoter: pl6INK4a, ERa, Ecadherin. Based on sequence knowledge and in silico theoretical bisulphite reactions modifications, four probes were designed for each CpG evaluation. These probes correspond to the four possible different results from being subjected to the method of the invention. These situations correspond to forward and reverse directions, with and without complete cytosine methylation (see FIG. 3).
  • PCR primers used to obtain the promoter fragments were as follows.
  • ER ⁇ -F CAGCAGCGACGACAAGTAAA (SEQ ID NO:1)
  • ER ⁇ -R TCCAAACACCCCCAACTTTA (SEQ ID NO:2)
  • Ecad_B_ F CTCACACCTGAAATCCTAGC (SEQ ID NO:3)
  • Ecad_B_R CCCTCAACCTCCTCTTCTTT (SEQ ID NO:4)
  • pl6INK4a _B_ F GCTCCTCATTCCTCTTCCTT (SEQ ID NO: 5)
  • pl6INK4a _B'_R TCCCTCCCCATTTTCCTATC (SEQ ID NO:6) PCR was performed using 30 ng DNA genomic template and Taq
  • the PCR mix consisted in 1 ⁇ l of genomic DNA, 1.5 mM MgCl 2 , 200 nM dNTP, 4% DMSO, 1 x Buffer Taq , 0.25 pmol forward primer, 0.25 pmol reverse primer and 1 U Taq Polymerase.
  • the PCR protocol consisted of a denaturing step of three minutes at 94 0 C, followed by 34 cycles of 94 0 C for one minute, 58 0 C for one minute, and 72 0 C for two minutes with a final extension at 72°C for ten minutes.
  • the PCR conditions were a denaturing step of three minutes at 94 0 C, followed by seven cycles at (94 0 C for one minute, 64 0 C for one minute, and 72 0 C for two minutes) followed by 24 cycles of 94°C for one minute, 57°C for one minute, and 72°C for two minutes with a final extension at 72°C for ten minutes.
  • PCR products were purified from primers and dNTPs by Amicon Microcon-PCR Centrifugal Filter Devices (UFC7 PCR 50, Millipore, Bedford, MA, USA).
  • the ends of the cleaved DNA fragments were ligated to phosphorylated and cytosine-methylated adaptors.
  • the ligation-mixture with 1600 ng promoter regions was supplemented with 3 ⁇ l of 10 x ligation buffer (Fermentas), 1 ⁇ l T4 Ligase (Fermentas) (5 U), 20:1 ratio DNA:adapter pmols and water to 30 ⁇ l.
  • the ligation reaction was carried out at room temperature for 3.5 hours.
  • the EZ DNA methylation kit (Zymo Research) was used for bisulphite conversion of all promoter samples used in this study, according to the manufacturer's recommendations. For each conversion, 500 ng of ligated DNA was used. The complementary strands of the bisulphite treated sDNA were synthesized with a primer extension, using a T7 forward primer, the sequence of which was contained in one of the adaptor.
  • This template double strand DNA was in vitro transcribed to RNA with 40 U T7 RNA Polymerase (Ambion), 7,5 mM rNTPs, 2 ⁇ l Cy3-CTP 6 mM (PerkinElmer,
  • RNA (corresponding to methylated) with 100 ng RNA (corresponding to unmethylated) for each hybridization were pooled with 11 ⁇ l of Blocking Agent (Agilent), 2.2 ⁇ l of 25 x Fragmentation Buffer (Agilent, Santa Clara, California) and nuclease-free water to 52.8 ⁇ l. Before hybridization to the array, the samples are incubated 30 minutes at 65°C to fragment RNA. Then, 55 ⁇ l of 2x GExHybridization Buffer Hi-rpm (Agilent) was added to stop the fragmentation reaction.
  • Blocking Agent Agilent
  • Fragmentation Buffer Agilent, Santa Clara, California
  • the sample was applied to the array by using Agilent microarray hybridization chamber, and hybridization was carry out for 17 hours at 65°C in a rotating oven at 10 rpm.
  • the arrays were then disassembled in Gene Expression Wash Buffer 1 (Agilent) then washed with Wash Buffer 1 and 2 (Agilent).
  • the slides were dried with acetonitrile wash and stabilization and drying solution according to manufacturer's recommendations. Slides were scanned by using and Agilent 62505B DNA microarray scanner.
  • FIGS. 4A, 4B, and 4C show co-hybridization scatter-plots for the three promoters corresponding to the completely not methylated vs. completely-methylated samples.
  • a second chip was designed as described in Example 1 but containing probes for a larger set of promoters: pl6INK4a, ERa, Ecadherin, MGMT, GSTPl, and APC.
  • Genomic DNA extracted from a clinical sample (2 ⁇ g) was incubated with Taql (Fermentas Canada, Burlington, Ontario) (10 U) in a total volume of 30 ⁇ L overnight at 37°C to give digested genomic DNA.
  • Taql Fermentas Canada, Burlington, Ontario
  • the ends of the cleaved DNA fragments were ligated to phosphorylated and cytosine-methylated adaptors.
  • the ligation-mixture with 1600 ng DNA genomic was supplemented with 3 ⁇ l of 10 x ligation buffer (Fermentas), 1 ⁇ l T4 Ligase
  • the EZ DNA methylation kit (Zymo Research) was used for bisulphite conversion of all promoter samples used in this study, according to the manufacturer's recommendations. For each conversion, 500 ng of ligated DNA was used.
  • the complementary strand of the bisulphite treated sDNA was synthesized with a primer extension reaction, using a T7 forward primer, which sequence was contained in one adaptor.
  • the template double strand DNA was in vitro transcribed to RNA with 40 U T7 RNA Polymerase (Ambion), 7.5 mM rNTPs, 2 ⁇ l Cy3-CTP 6 mM (PerkinElmer) or 2 ⁇ l Cy5-CTP 4 mM (PerkinElmer) overnight at 37 0 C.
  • RNA samples were purified with MEGAclearTM columns (Ambion). 100 ng methylated RNA with 100 ng unmethylated RNA for each hybridization were pooled with 11 ⁇ l of Blocking Agent (Agilent), 2.2 ⁇ l of 25 x Fragmentation Buffer (Agilent) and nuclease-free water to 52.8 ⁇ l. Before hybridization to the array, the samples were incubated 30 minutes at 65°C to fragment the RNA. Then, 55 ⁇ l of 2x GExHybridization Buffer Hi-rpm (Agilent) are added to stop the fragmentation reaction.
  • Blocking Agent Agilent
  • Fragmentation Buffer Fragmentation Buffer
  • the sample was applied to the array by using Agilent microarray hybridization chamber, and hybridization was carry out for 17 hours at 65°C in a rotating oven at 10 rpm.
  • the arrays were then disassembled in Gene Expression Wash Buffer 1 (Agilent) then washed with Wash Buffer 1 and 2 (Agilent).
  • the slides were dried with acetonitrile wash and stabilization and drying solution according to manufacturer's recommendations. Slides were scanned by using and Agilent 62505B DNA microarray scanner.
  • FIG. 5 shows high specificity of the methodology in the identification of the methylation state of each promoter sequence in a human complex clinical sample: probes specifically designed for methylated sequence give significant positive response just for artificial methylated samples and probes specifically designed for the completely not methylated sequence gave major significant positive response for completely not methylated samples (as the six promoters of tumor suppressor genes have been previously described to be unmethylated in healthy human samples).

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Abstract

L'invention porte sur des procédés et des compositions utiles pour détecter la méthylation d'ADN pour, par exemple, caractériser l'état de méthylation ou le profil de méthylation d'ADN. Les procédés peuvent être utilisés pour étudier l'état/le profil de méthylation d'un promoteur spécifique/îlot CpG spécifique et/ou d'une pluralité de promoteurs/îlots CpG. Les compositions peuvent être utilisées pour évaluer la méthylation d'ADN de l'ADN génomique. Les procédés sont utiles dans diverses applications comprenant le diagnostic et le pronostic de maladies présentant des motifs de méthylation d'ADN modifiés ou pour la découverte de biomarqueurs. L'invention peut être utilisée pour identifier des biomarqueurs spécifiques associés à des phénotypes et pour établir des empreintes de méthylation (par exemple des motifs, des états, des profils ou le méthylome). Les motifs de méthylation, états de méthylation, profils de méthylation et le méthylome tels que déterminés par les procédés peuvent être associés à des phénotypes (pronostic, diagnostic, réponse à des agents thérapeutiques, etc.). Les procédés et compositions sont en outre utiles pour déterminer des motifs de méthylation à l'échelle du génome.
PCT/EP2009/002003 2008-03-19 2009-03-18 Procédé et composition pour analyse de méthylation Ceased WO2009115313A1 (fr)

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US10900064B2 (en) 2011-09-01 2021-01-26 Belgian Volition Sprl Method for detecting nucleosomes containing nucleotides
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CN118497334A (zh) * 2024-06-05 2024-08-16 武汉大学中南医院 抑制flnc基因表达的物质在制备治疗良性前列腺增生的药物中的应用
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9632049B2 (en) 2009-12-07 2017-04-25 Ams International Ag Integrated circuit and manufacturing method therefor
US10900064B2 (en) 2011-09-01 2021-01-26 Belgian Volition Sprl Method for detecting nucleosomes containing nucleotides
US12529107B2 (en) 2016-11-07 2026-01-20 University Of Cincinnati CpG island methylation profile in non-invasive oral rinse samples for detection of oral and pharyngeal carcinoma
CN113584168A (zh) * 2021-07-19 2021-11-02 深圳泰莱生物科技有限公司 基于甲基化免疫沉淀高通量测序技术的肺癌检测方法
CN118497334A (zh) * 2024-06-05 2024-08-16 武汉大学中南医院 抑制flnc基因表达的物质在制备治疗良性前列腺增生的药物中的应用

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