WO2008135512A2 - Procédé d'amplification de l'adn - Google Patents

Procédé d'amplification de l'adn Download PDF

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Publication number
WO2008135512A2
WO2008135512A2 PCT/EP2008/055377 EP2008055377W WO2008135512A2 WO 2008135512 A2 WO2008135512 A2 WO 2008135512A2 EP 2008055377 W EP2008055377 W EP 2008055377W WO 2008135512 A2 WO2008135512 A2 WO 2008135512A2
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dna
nucleotides
methyiation
polynucleotide
profile
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PCT/EP2008/055377
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WO2008135512A3 (fr
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Jerzy Paszkowski
Jon Reinders
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Jerzy Paszkowski
Jon Reinders
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Publication of WO2008135512A2 publication Critical patent/WO2008135512A2/fr
Publication of WO2008135512A3 publication Critical patent/WO2008135512A3/fr

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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 present invention is in the area of DNA methylation profiling.
  • the invention relates to a method of using bisuifite-mediated cytosine conversion, followed by random amplification using a unique primer design, and processing for arrays or other high- throughput sequence analysis methods.
  • the method according to the invention allows improved detection sensitivity and high resolution over the current state of the art of genome-wide detection of DNA methylation,
  • DNA methylation is found in wide array of organisms ranging from bacteria to humans. In general, DNA methylation is catalyzed by DNA methyltransferases adding a methyl group to the N6 position of adenine or the C5 or N4 position of cytosine.
  • the main focus within the scope of the present invention is on the impact and detection of cytosine methylation at the CS position (SmC), the predominantly modified base in multicellular eukaryotes,
  • DNA methyiation is imprinting of silenced promoters on the inactive X chromosome , but more recently a 2-fold increase of allele specific DNA methyiation within genes on the active X versus the inactive X chromosome indicates methylation is not repressive within all genomic positions 2 . Indeed, reports of body methyiation within actively expressed genes were reported in Arabidopsis 3 4 5 , Similarly, the importance of DNA rnethylation during mammalian development for correct embryonic gene expression and development has been appreciated, but has recently gained additional interest towards developing stem cell therapies . Another major research interest regarding the role of DNA methylation has resulted from understanding that DNA methylation in cancer cells can be different from normal cells 7 .
  • MSREs methylation-sensit ⁇ ve restriction enzymes
  • Cytosine conversion upon sodium bisulfite treatment was first reported in 1970 15 , but the major advancement for DNA methyiation analysis occurred upon the advent of PCR techniques 16 . Briefly, 5-methyicytosine detection is based on the specific reaction of bisulfite with cytosine, which is converted to uracil after subsequent alkaline hydrolysis. Following PCR amplification, the uracil corresponds to thymidine at the converted site 17 . However, 5-methylcytosine is not modified under these conditions. Thus, bisulf ⁇ te-mediated cytosine conversion identifies C-to-T transitions, analogous to single nucleotide polymorphisms (SNPs), to allow detection of methylation polymorphisms.
  • SNPs single nucleotide polymorphisms
  • the invention provides a method for amplification of a genomic polynucleotide sample comprising a polynucleotide treated with a modifying agent that modifies unmethyiated cytosine to produce a converted nucleic acid, particularly a sodium bisulfite-treated polynucleotide, which method comprises a. contacting a genomic polynucleotide sample with a modifying agent, particularly a sodium bisulfite, that modifies unmethyiated cytosine to produce a converted nucleic acid, b.
  • a modifying agent particularly a sodium bisulfite
  • a first amplification round amplifying said polynucleotide sample by means of a first oligonucleotide primer comprising essentially two portions, wherein the first portion at its 3' end comprises a random nucleotide sequence, particularly a sequence of less than 6 random nucleotides, and the second portion at its 5' end comprises a defined nucleotide sequence, particularly a sequence of at least 10 defined nucleotides, even more particularly of at least 15 defined, but especially of at least 20 defined nucleotides and up to at least 30-40 defined nucleotides; and c. in a second amplification round amplifying the first round primary amplification product by means of a second oligonucleotide primer comprising essentially only the second defined portion of the first oligonucleotide primer used in step b..
  • the polynucleotide sample is a DNA sample. In one embodiment of the invention, the polynucleotide sample is a RNA sample.
  • the polynucleotide is from an animal, particularly a mammal, but especially a human.
  • the polynucleotide is from a plant, particularly a plant of agronomic importance such as a field crop or a vegetable plant.
  • the genomic DNA is digested for sample preparation using restriction enzymes, particularly enzymes lacking cytosine residues within the recognition site.
  • the digestion products are 30Kb or less, particularly the digestion products are in a range of between 0.5-10Kb and more particularly 5 Kb.
  • the digested DNA is treated with sodium bisulfite such as to convert essentially ail non-methylated cytosines to uracil, and to maintain all methylated cytosines as cytosines; and to minimize fragmentation effects due to depurination during the treatment.
  • sodium bisulfite such as to convert essentially ail non-methylated cytosines to uracil, and to maintain all methylated cytosines as cytosines; and to minimize fragmentation effects due to depurination during the treatment.
  • UDG uracil DNA glycosylase
  • the sodium bisulfite and sodium hydroxide molecules are efficiently removed by the end of the treatment.
  • the 3' random portion of the first oligonucleotide primer comprises between 3 - 5 random nucleotides, particularly 4 random nucleotides and the defined portion between 15 - 30 defined nucleotides, particularly between 15 - 20 defined nucleotides. It is aiso within the spirit of the current invention that alternative approaches using adaptor molecules could be ligated to the DNA using techniques known to those skilled in the art. Such modifications would incorporate a known primer sequence before, during or after, the bisulfite conversion, or the amplification process, to facilitate "universal" PCR amplification conditions and to confer specificity of the amplification process for amplification of only converted DNA template.
  • the random nucleotides can be any of the nucleotides, for example G, A, T or C in any order, wherein: G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidyiic nucleotides and C cytidylic nucleotides.
  • the first oligonucleotide primer to be used in the method according to the invention may contain ai! combinations of these nucleotides in every position of the random portion of the primer.
  • the 3' end of the primer wilf be complementary to random sites throughout the target DNA segments.
  • the invention relates to a method of amplifying bisuifite-treated DNA according to the invention, wherein in first round amplification a. a primary reaction mixture is provided comprising: (i) a bisuifite-treated DNA sample;
  • a first oligonucleotide primer according to the invention and as described herein above comprising essentially two portions, wherein the first portion at its
  • 3' end comprises a random nucleotide sequence, particularly a random 4-mer sequence of nucleotides and the second portion at its 5' end comprises a defined nucleotide sequence, particularly a defined nucleotide sequence of between 15 -20 nucleotides of a known sequence 5' of the random nucleotides;
  • a mixture of nucleotides dNTPs
  • dNTPs nucleotides
  • G is understood to represent guanylic nucleotides
  • A adenylic nucleotides, T thymidyiic nucleotides and C cytidylic nucleotides, which nucleotides may be labeled, particularly with a fluorescence marker
  • a DNA polymerase, or combination of multiple DNA polymerases particularly a heat sensitive DNA polymerase exhibiting primer displacement activity but essentially no exonuclease activity, more particularly a T7 DNA polymerase, but especially a modified T7 DNA polymerase such as, for example, a Sequenase or Phage Phi29 DNA polymerase possessing primer displacement activity that results in longer polymerization products.
  • the first round amplification comprises at least a second amplification cycle comprising the steps of denaturing the primary DNA amplification product; reannealing the first oligonucleotide primer with the DNA to aliow the formation of a DNA-p ⁇ mer hybrid; and allowing the heat sensitive DNA polymerase to extend synthesis from the DNA-primer hybrids formed within each cycle to produce DNA fragments comprising the DNA segment to be amplified which is flanked by the defined sequence of the first oligonucleotide primer on their 5 1 ends and the reverse complement of that defined sequence on their 3' ends.
  • This amplification cycle is repeated at least once, but may be repeated for a total of between 2 to 8 times, particularly for a total of between 3 and 6 times, wherein fresh enzyme is to be added for each cycle.
  • the invention relates to a method of amplifying bisulfite- treated DNA according to the invention, wherein in second round amplification b. a secondary reaction mixture is provided comprising
  • dNTPs nucleotides
  • G is understood to represent guanylic nucleotides
  • DNA polymerase particuiariy a heat-stabSe DNA polymerase, but especially a
  • Taq DNA polymerase This method is specifically suitable for preparation of samples to be hybridized to high density oligonucleotide arrays.
  • the invention in still another specific embodiment, relates to a method of amplifying bisuifite-treated DNA according to the invention, wherein in second round amplification c. a secondary reaction mixture is provided comprising
  • a second primer comprising the defined sequence portion at the 5' end of the first primer but lacking the random sequence of nucleotides at its 3 1 end;
  • a mixture of nucleotides dNTPs), particuiariy a mixture of dATP, dCTP, dGTP, dTTP, wherein G is understood to represent guanyiic nucleotides, A adenylic nucleotides, T thymidylic nucleotides, and C cytidyiic nucleotides, which nucleotides may be labeled, particularly with a fluorescence marker; (xii) DNA polymerase, particularly a heat-stable DNA polymerase, but especially a
  • the second round of DNA amplification comprises the steps of denaturing the primary DMA product before annealing the second primer with the DNA product to allow the formation of a DNA-primer hybrid; and incubating the DNA-primer hybrid to allow the heat-stable DNA polymerase to synthesize a second DNA product.
  • the invention provides a method for detecting methySation patterns within a genomic polynucleotide sample, particularly a genomic DNA sample, comprising: a. contacting a genomic polynucleotide sample with a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid; b, in a first amplification step treating said polynucleotide sample by means of an oligonucleotide primer comprising essentially two portions, wherein the first portion at its 3' end comprises a random nucleotide sequence, particularly a sequence of less than 6 random nucleotides, more particuiarly of between 2 and 5 random nucleotides, even more particularly of between 3 and 5 random nucleotides, but especially of 4 random nucleotides, and the second portion comprises a defined nucleotide sequence, particuiarly a sequence of at least 5 defined nucleotides, more particularly of at ieast 10 defined nucleotides, even
  • a second amplification round amplifying the first round primary amplification product by means of a second oligonucleotide primer, comprising essentially only the second defined portion of the first oiigonucieotide primer used in step b); and d. using said secondary amplification product for detecting cytosine methylation and methylated CpG islands in said polynucleotide sample.
  • the polynucleotide sample is a DNA sample. In one embodiment of the invention, the polynucleotide sample is a RNA sample.
  • the polynucleotide is from an animal, particularly a mammal, but especially a human.
  • the polynucleotide is from a plant, particularly a plant of agronomic importance such as a field crop or a vegetable plant.
  • the invention provides a method for detecting methylation patterns within a genomic sample of DNA according to the present invention, wherein in first round amplification a. a primary reaction mixture is provided comprising: (i) the converted DNA sample produced in step a);
  • a first oligonucleotide primer comprising essentially two portions, wherein the first portion at its 3' end comprises a random nucleotide sequence, particularly a random 4-mer sequence of nucleotides and the second portion at its 5' end comprises a defined nucleotide sequence, particularly a defined nucleotide sequence of between 15 - 20 nucleotides of a known sequence 5 1 of the random nucleotides;
  • dNTPs a mixture of nucleotides
  • G is understood to represent guanyiic nucleotides, A adenylic nucleotides, T thymidyfic nucleotides and C cytidylic nucleotides
  • a DNA polymerase particularly a heat sensitive DNA polymerase exhibiting primer displacement activity but essentially no exonuciease activity, more particularly a T7 DNA
  • the DNA sample is denatured; c. step b) is repeated in a second amplification cycle allowing the random segments of the first oligonucleotide primer to anneal to their complementary sequences; and d. using said secondary amplification product for detecting cytosine methylation and methylated CpG islands in said polynucleotide sample.
  • the invention provides a method for detecting methyiation patterns within a genomic sample of DNA according to the present invention, wherein in second round amplification a. a secondary reaction mixture is provided comprising:
  • dNTPs nucleotides
  • G is understood to represent guanyiic nucleotides
  • a DNA polymerase particularly a heat-stable DNA polymerase, more particularly a Taq polymerase.
  • the invention provides a method for detecting methylation patterns within a genomic sample of DNA according to the present invention, wherein in the second round amplification ⁇ b. a secondary reaction mixture is provided comprising; (v) the primary reaction product; (vi) a second primer comprising the defined sequence portion at the 5 !
  • a mixture of nucleotides dNTPs
  • dNTPs mixture of nucleotides
  • G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidylic nucleotides, and C cytidylic nucleotides
  • a heat-stable DNA polymerase particularly a heat-stable DNA polymerase, more particularly a Taq polymerase
  • the polynucleotide amplified in a method according to the invention and as described herein before can be used in a method for identifying methylation polymorphisms, which method is selected from the group consisting of (a) conventional, locus-specific PCR-based cloning; (b) quantitative PCR detection, including pyrosequencing approaches; (c) random "shotgun" genome sequencing approaches; (d) ultra high-throughput picoliter pyrosequencing techniques; (e) ultra high-throughput massively parallel signature sequencing (MPSS), sequencing by synthesis (SBS), or clonal single molecule array (CSlVlA); (f) "bead array” technologies using silica beads that self assemble in microwells on a substrate(s), (g) allelic discrimination assays using either single base extension of 5 !
  • exonuc!ease reporter probe (Taqman) assays, including microfluidic "card” formats, barcode chips, or bioeiectronic chips, and (h) design of methylation-specific oligonucleotide arrays or microfluidic "card” formats derived from i ⁇ - siiico conversion of sequences detected as DNA methylation polymorphisms identified by hybridizaton of the amplified DNA to standard, commercially available oligonucleotide arrays or custom-designed "spotted” cDNA arrays.
  • the amplified polynucleotide can be applied towards conventional, locus-specific PCR-based cloning.
  • the method according to the invention and as described herein before can be applied towards either direct sequencing of the amplified polynucleotide for detection of methylation patterns, particularly methylation polymorphisms.
  • the amplified polynucleotide, particularly the amplified DNA can be analyzed using standard cloning techniques known to those skilled in the art. 32
  • the amplified polynucleotide can be applied towards quantitative PCR.
  • state of the art methods such as those described in US Patent 7,112,404 and US Patent 6,331 ,393, herein incorporated by reference, which currently use not amplified sodium bisulfite-treated DNA directly for detecting methylation patterns, particularly methylation polymorphisms, may be applied to polynucleotides amplified according to the present invention.
  • the amplified DNA obtainable in a process according to the present invention may be analyzed using a related quantitative PCR method involving energy transfer of a "beacon probe" that can form hairpin structures as described in US Patent 5,119,801 and US Patent 5,312,728, respectively, herein incorporated as references.
  • a "beacon probe” On one end of the hybridization probe (either the 5 1 or 3' end), there is a donor fiuorophore, and on the other end, an acceptor moiety.
  • the molecuiar beacon probe can hybridize to one of the strands of the PCR product, allowing an "open conformation," and fluorescence detection, while non-hybridized beacon probes do not fluoresce 22 30 .
  • the fluorescence signal intensity may be used to quantitatively measure the abundance of methylation polymorphisms.
  • amplified DNA obtainable in a process according to the present invention may be used for detecting methylation patterns, particularly methylation polymorphisms by applying a method as described in US Patent 7,037,650, herein incorporated by reference, where amplified DNA resulting from the current invention would have strand-specific primer binding sites for PCR amplification and then for performing a primer extension reaction, or "Ms-SNuPE", The resulting fluorescent signal can determine the methylation state at the first primer-extended base.
  • amplified DNA obtainable in a process according to the present invention may be used for the 5' nuclease PCR assay, referred to as a "Taqfvla ⁇ " assay 31 according to the previously described method.
  • the amplifying DNA obtainable in a process according to the present invention may be used in sequencing techniques, including but not limited to: for random "shotgun" genome sequencing, high-throughput picoliter pyrosequ ⁇ ncing, massively parallel signature sequencing (MPSS), sequencing by synthesis (SBS) 1 or clonal single molecule array (CSMA).
  • sequencing techniques including but not limited to: for random "shotgun" genome sequencing, high-throughput picoliter pyrosequ ⁇ ncing, massively parallel signature sequencing (MPSS), sequencing by synthesis (SBS) 1 or clonal single molecule array (CSMA).
  • MPSS massively parallel signature sequencing
  • SBS sequencing by synthesis
  • CSMA clonal single molecule array
  • enrichment of methylated DNA using the aforementioned stategies including but not limited to using methyi binding domain proteins coupled with affinity chromatography or anti ⁇ 5mC antibodies to immunoprecipitate methylated DNA 21 28 3 4 , followed by bisulfite treatment and amplification of the DNA in a
  • the amplified DNA obtainabie in a process according to the present invention may be used in "bead array” technologies using silica beads that self assemble in microwells on a substrate(s), such as fiber optic bundles or planar silica slides, with uniform spacing on said substrate, and each bead is covered with many copies of a specific oligonucleotide that hybridize complementing sequences.
  • the amplified DNA obtainable in a process according to the present invention may be used in a method to identify methylation polymorphisms according the method described in US Patent 6,977,146, herein incorporated by reference.
  • the amplified DNA obtainable in a process according to the present invention may be used as input for hybridization to a set of probes of different nucleobase sequences where the non-hybridized probes are separated and the hybridized probes are analyzed in a mass spectrometer. Assignment of the peak pattern obtained from the mass spectra to the methylation pattern and comparison of the new data with a database can lead to detection of methyfation polymorphisms.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards the identification of markers for diagnostic or prognostic evaluation of samples for associations to disease conditions, notably cancer.
  • the methylation polymorphisms may be used for determining whether a treatment or therapeutic agent, or combination therein, will be most likely to effectively treat the condition or for selecting alternative treatment options.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards the identification of epigenetic modifications associated with epigenetic reprogramming, developmental differentiation, or differentiation.
  • the process of the current invention could for DNA samples extracted from an organelle, single ceil, tissue, or organ sampled during these steps to detect deveJopmentally associated methylation poiymorphisms.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards methylation profiling with copy number variation within the nucleic acid to determine the association between the methylation of the nucleic acid and the effect of structural variation in the nucleic acid.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards comparing the DNA methylation profile of the nucleic acid in response to changes in the ploidy level of an organism, including, but not limited to: (a) autotetrapioidy, (b) a ⁇ otetraploidy, and (c) aneuploidy to determine the association between the DNA methylation state and the organism's ploidy level.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards comparing the DNA methylation profile of the nucleic acid in response to changes in the allelic composition of an organism, including, but not limited to: (a) to assess the impact of DNA methylation on the relative fitness of first generation progeny, e.g. hybrid vigor (heterosis), (b) subsequently self-fertiiized generations resultant from a cross-fertilization to assess the impact of DNA methylation on phenotypic variation or relative fitness, and (c) back-crossed populations to determine the association between the DNA methylation state and the organism's fitness level.
  • first generation progeny e.g. hybrid vigor (heterosis)
  • subsequently self-fertiiized generations resultant from a cross-fertilization to assess the impact of DNA methylation on phenotypic variation or relative fitness
  • back-crossed populations to determine the association between the DNA methylation state and the organism'
  • the amplified DNA obtainable in a process according to the present invention can be applied towards comparing the methyiation profile before, during or after stem cell therapy treatments for either diagnostic or prognostic purposes.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards comparing the methylation profiie before, during or after RNAi treatments, including, but not limited to the delivery of siRNA, miRNA, piRNA, rasiRNA, nat-siRNA, tran siRNA or other small RNA molecules into a specimen or patient.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards comparing the methylation profiie before, during or after gametogenesis of a specimen or patient for either diagnostic or prognostic purposes.
  • the amplified DNA obtainable in a process according to the present invention can be applied towards for the diagnosis and/or prognosis of adverse events for patients or individuals, whereby these adverse events belong to any of the following categories: u ⁇ desired drug interactions; cancer diseases; central nervous system malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinicai, psychological and social consequences of brain damage; psychotic disturbances and personality disorders; dementia; cardiovascular disease, malfunction and damage; malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; iesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disease of the skin, of the muscles, of the connective tissue or of the bones; endocrine and metabolic malfunction, damage or disease; headaches or sexual malfunction.
  • adverse events belong to any of the following categories: u ⁇ desired drug interactions; cancer diseases; central nervous system malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinicai, psychological and
  • FIG. 1 Overview of the amplification method
  • BiMP Bisulfite Methylation Profiling
  • probe sequences Upon hybridization, highly converted probe sequences will fail to complement the array features, even though such probes are labeled and present, resulting in low or absent fluorescent signal intensities at such features (purple array feature). Probes retaining partial sequence complementarity will hybridize with an intermediate signal intensity (blue array feature). Probes retaining high sequence complementarity will hybridize with a high signal intensity (green array feature), indicated by the bright red fluorescence. The resulting fluorescent signal intensities per array feature then infer the original DNA methyiation patterns of the corresponding sequence.
  • FIG. 1 Graphical representation of the Bisulfite Methyiation Profiling (BiMP) amplification method
  • converted, single-stranded DNA molecules are mixed with the oligonucleotide primer comprised of two portions: a 5 1 region comprised of a defined nucleotide sequence and the 3' region comprised of a random nucleotide sequence, such as 4 random nucleotides.
  • random annealing occurs (a) followed by elongation to complete synthesis of the first strand (b).
  • the double-stranded DNA templates are denatured and the steps are repeated, thus incorporating sequences complementing the known primer sequence (c).
  • step two the newly synthesized double-stranded DMA templates are amplified using PCR in the presence of dNTP, including dUTP to facilitate fragmentation.
  • a cytosine conversion method is used to generate methyiation profiles on a high density oligonucleotide array that result in detection sensitivity at the 35 bp resolution 34 ( Figure 1 ),
  • the invention relates to a method for detecting cytosine methylation and methylated CpG islands within a genomic polynucleotide sample comprising: a. contacting a genomic polynucleotide sample with a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid, b.
  • a first amplification round amplifying said polynucleotide sample by means of a first oligonucleotide primer comprising essentially two portions, wherein the first portion at its 3' end comprises a random nucleotide sequence, particularly a sequence of less than 6 random nucleotides, and the second portion at its 5' end comprises a defined nucleotide sequence, particularly a sequence of at least 10 defined nucleotides, c. in a second amplification round amplifying the first round primary amplification product by means of a second oligonucleotide primer comprising essentially only the second defined portion of the first oligonucleotide primer used in step b); and d. using said secondary amplification product for detecting methylation polymorphisms,
  • the DNA to be used in the method according to the invention is preferably pure DNA without detectable RNA or protein contamination. It is further preferred to apply known measures to ensure complete conversion. It is further preferred to digest high molecular weight genomic DNA into smaller fragments, particularly into fragments of 30Kb or less, to ensure the DNA molecules remain as single strands that can be modified during sodium bisulfite treatment. It is not advisable to sonicate or nebulize the DNA, These treatments are inhibitory to DNA conversion, possibly due to DNA damage caused hydroxy! radicals generated during cavitation or end damage caused during shearing. It is preferred to completely remove the sodium bisulfite sails prior to the alkali desuiphonation step.
  • the fragmentation caused by depurination is limited using known methods.
  • a commercially available bisulfite conversion kit may be used containing a DNA protection buffer. However, controlling these parameters alone is insufficient for unbiased probe ampiification.
  • Bisuifite-treated DNA amplification can further to be improved by modulating the DNA polymerase concentrations, dNTP composition, and particularly by providing a new primer design ( Figure 2).
  • a novel primer design is introduced to compensate for any negative degradation effects or negative base pair composition effects inherent to the bisulfite amplification process. Comparing DNA amplification products amplified using the known and novel methods does not result in visually detectable differences observed using gel electrophoresis (data not shown ⁇ . However, the sample integrity can be assayed using either locus specific PCR or dot blot analysis to identify amplification bias.
  • the improved amplification fidelity of the novel amplification method is supported by more consistent post-amplification PCR results (see Reinders et a/, Fig.
  • the amplification method according to the invention is thus especially suitable for preparing samples for hybridizations to polynucleotide arrays such as, for example, high density tiling arrays.
  • the primer according to the invention comprises essentially two portions, wherein the first portion at the 3' end of the primer comprises a random nucleotide sequence, particularly a random nucleotide sequence consisting of less than 6 nucleotides, more particularly a random nucleotide sequence consisting of between 3 - 5 nucleotides, but especially a 4-mer sequence of nucleotides and the second portion at its 5 1 end of the primer comprises a defined nucleotide sequence, particularly a defined nucleotide sequence of between 15 - 20 nucleotides of a known sequence 5' of the random nucleotides ( Figure 2).
  • the method according to the present invention incorporated a DNA reaction mixture, which is comprised of a bisuifite-treated DNA, a first oligonucleotide primer, and a reaction buffer.
  • the components to this reaction mixture are mixed and heat denatured, particularly for about 2 minutes at about 94°C.
  • the sample is cooled, particularly to about 4 to 10 0 C at a rate of about 2°C s "1 , such that the random segments of the primers anneal to complementary sequences.
  • the complementary sequences occur randomly on the bisulfite-treated DNA segments.
  • the polymerase reaction mix comprising DNA polymerase buffer, dATP, dCTP, dGTP, dTTP, DTT (dithiothreitol), bovine serum albumin (BSA), and the DNA polymerase is prepared and added to the DNA reaction mixture and gently mixed.
  • the preferred DNA polymerase used in this step is a heat sensitive enzyme with primer dispfacement activity and without exonuciease activity.
  • the polymerase used is preferably a T7 DNA polymerase and more preferably a modified T7 DNA polymerase (Sequenase Version 2.0; United States Biochemicals), possessing primer displacement activity that results in longer polymerization products.
  • the temperature is increased to about 37C at a rate of about 0.05C/second and maintained for roughly eight minutes allowing polymerase extension to occur for synthesis of DNA segments with the known sequence of the primer at their 5' ends.
  • This reaction mix is then reheated for denaturation and primers are re-annealed.
  • fresh enzyme prepared in the polymerase buffer preferably the Sequenase dilution buffer, is added since the reheating process denatures the polymerase. This cycle is repeated at least one time, but may be repeated more.
  • the first primer wili anneal to first cycle reaction products resulting in amplified DNA flanked by the defined sequence of the primer on their 5" end and the reverse complement of that sequence on the 3 1 end.
  • the final reaction products of the primary ampiification cycles may or may not be diluted using distilled, sterile water or high-performance liquid chromatography (HPLC)-purified water, ranging from between 1- to 100-fold, particularly from between 2- to 20-foid, more particularly about fivefold, Other diiutio ⁇ factors may be acceptable with a particular set of reactions and is encompassed by the present invention.
  • the DNA amplification reaction comprising the newly synthesized DNA template, resulting from the primary reactions and described above, magnesium chloride, PCR amplification buffer, dATP, dCTP, dGTP, dTTP, and, optionally, dUTP, a second oligonucleotide primer comprising essentially only the second defined portion of the first oligonucleotide primer used in the primary reactions, water, and a DNA polymerase, preferably Taq DNA polymerase is amplified.
  • a DNA polymerase preferably Taq DNA polymerase
  • dUTP is incorporated into the reaction mixture of the secondary reactions, which may be advantageous for certain downstream application, such as, for example, for enzymatic fragmentation.
  • the ratio between dTTP and dUTP can vary according the desired fragment size, where increased dUTP wili generate more fragmentation sites resulting in shorter DNA fragments. Such an effect may increase the signal differences between methylated and unmethylated templates, leading to improved signal detection of methylation polymorphisms. Alternatively, less dUTP incorporation equates into few fragmentation sites, resulting in longer fragment sizes.
  • the ratio between dTTP and dUTP is in a range of between 1 :1 to 6:1 , particularly in a range of between 2:1 to 5:1, but especiaiiy 4:1.
  • the reaction is heated to about 94 0 C from about thirty seconds to about 4 minutes, preferably for about 3 minutes to denature the DNA.
  • the DNA is then amplified using thermal cycling conditions comprising a 30 second heat-denaturing step at about 94 0 C, a
  • PCR can be used and are we!! known to those of skill in the art and that certain reactions may require specific proportions of the various reaction components concentration.
  • the secondary amplification products may then be subjected to fragmentation procedures such as, for example, uracil DNA glycosylase (UDG) and apurinic/apyrimidintc endo ⁇ ucJease 1 (APE 1) mediated fragmentation.
  • UDG uracil DNA glycosylase
  • APE 1 apurinic/apyrimidintc endo ⁇ ucJease 1
  • a modified protocol is used to guarantee complete fragmentation of the amplified DNA and thus reproducibility of the method since it affects the likelihood that the labeled target can globally hybridize evenly across the array.
  • Fragmentation conditions are modified such as to obtain a population of fragmented DMA with an average size of approximately 66bp.
  • amplified DNA is treated under conditions as previously reported (Affymetrix), but with the modification that treatment was performed for about 120 minutes at about 37 C C, followed by about 2 minutes incubation above 92 0 C 1 preferably at 94°C, and then cooling to about 4°C. Fragmentation can be evaluated using an Agilent Bioanaiyzer 2100 with the Eukaryote Total RNA nano assay suitable for detecting single stranded nucleic acids (see Reinders et a/, Supp Fig. 1 ). In an alternative embodiment, the amplified DNA is treated for 1 hour treatment but with higher enzyme concentrations.
  • the samples are prepared for hybridization on high density oligonucleotide arrays,
  • the fragmented DNA is preferably labeled with a detectable probe, particularly the fragmented DNA is end-labeled with a fluorescent marker such as a biotin.
  • fragmented DNA is labeled in a TdT buffered reaction using terminal deoxynucleotidy! transferase (TdT) in the presence of a bioti ⁇ ylated compound.
  • TdT terminal deoxynucleotidy! transferase
  • kits are available that may be used within the scope of the present invention, such as, for example, the GeneChip DNA Labeling Kit (Affymetrix ⁇ .
  • the labeling reaction is incubated for about 1 hour at about 37 ⁇ C, heated for about 10 minutes at about 70 0 C and then cooled for 2 to 10 minutes at about 4°C.
  • the labeled DNA is then added to hybridization cocktail comprised of a hybridization buffer, DMSO 1 B2 control oligo
  • RNA hybridization spikes 3 ⁇ m
  • water 3 ⁇ m
  • the hybridization mixture is then heated to about 94 0 C for about 5 minutes, cooled to about 45°C for about 5 minutes, and centrifuged at about 1300Og for about 5 minutes.
  • An aliquot of the reaction containing roughly 7.2 ⁇ g of labeled, fragmented DNA was hybridized for 16hours at 45°C.
  • the wash steps remove non-hybridized nucleic acids and the resulting signal intensity is a measure of the labeled probe hybridizing to the array features.
  • Methods for detecting complex formation are well known to those skilled in the art, incfuding confoca! fluorescence microscopy, argon ion laser excitation coupled to a photomultipiier for light quantification, or a computer-driven scanner device.
  • the resulting image can be examined to determine the abundance of each hybridized target polynucleotide and the data analyzed to determine the methylation patterns at the feature level
  • a microarray refers to a coated substrate, often glass slides or membrane filters, with high-density nucieSc acid samples, usually cDNA or oligonucleotides, which are delivered at discrete areas and immobilized to the substrate.
  • An array element herein referred to as a feature, refers to a place where an individual nucleic acid is located on the microarray.
  • a sample of fiuorescentiy fabeied nucleic acids (probe) is hybridized to the microarray.
  • the biological information may be about that sequence, such as genetic polymorphisms due to variant nucleotide composition or changes in mRNA transcript abundance, in the process according to the present invention, differential hybridization intensities infer cytosine conversions related to the initial cytosine methylation state borne on the nucleic acid ( Figure 1 ), herein referred to as a methylation polymorphism. It is within the spirit of the invention that altered stringency levels of the washing steps can affect hybridization intensities, thus allowing for optimization of this parameter to be suited for different specimens.
  • Microarray datasets can be normalized to ailow comparisons between multiple slides generated under similar test conditions.
  • Known methods include using the intensities from interna! controls or from the intensity of total genomic DNA hybridization.
  • Total genomic DNA hybridizations allow empirical determination of each feature's probe hybridization behavior and can be used as a reference control.
  • a reference control may be used to normalize between samples was amplified genomic DNA that accounted for probe hybridization behaviour variation and amplification bias inherent to the method. Examples
  • Example 1 DNA Extraction 1. Clean work area. Wipe off bench top to remove potential contaminating DNA, use
  • Entry 1 is CoI-O, the wild type Arabidopsis thaliana reference accession. Entry
  • the expected result is an increased proportion of probes originating from u ⁇ methylated DNA with lower fluorescent signal intensity in comparison to non treated DNA signal ( Figure 1).
  • Figure 1 To identify methylation polymorphisms, the wild type accession Co)-O, the reference Arabidopsis strain, and the null DNA methyitransferase mutant, met1-3 3B were used. This mutation causes a genome-wide loss of CpG methylation and significant reductions in non-CpG methylation 38 .
  • the hybridization results represented in a histogram distribution supports this expectation (see Reinders et a/, Supp Fig. 2).
  • methyiation polymorphisms at FWA and SUP are best documented and unequivocaiiy detected by BiMP, they were used to reveal novei DNA methyiation polymorphisms across the genome.
  • a positive cutoff level at 4.0 (representing a 16-fold signal difference) with a sliding window of 161bp was assigned, roughly the sequence length per nucleosome. Under these conditions, approximately 4% of the methyiation intensity differences between the entries were classified as significant
  • These methyiation polymorphisms consisted of 26,777 hypomethylated and 15,184 hypermethyiated intervals, representing approximately 2.7% (3,249,039bp) and 1.3% (1 ,533,464bp) of the array (1.19Mb), respectively.
  • the present invention for DNA methylation profiling has developed a novel approach to use the method of bisulfite-mediated cytosine conversion, followed by random amplification, using a unique primer design, to obtain DNA suitable for processing on high density tiling arrays or other high-throughput sequence analysts methods.
  • the method according to the invention allows improved detection sensitivity and higher resolution over the current state of the art of genome-wide DNA methylation profiling.
  • the application of this invention thus allows for improved detection of DNA methylation polymorphisms to generate high-resultion ep ⁇ genomic maps.
  • This result facilitates the ability to execute statistical associations between DNA methylation polymorphisms and phenotypes, using standard genetic approaches, including but not limited to: single- or multi-locus genetic mapping; quantitative trait locus (QTL) mapping; or association mapping approaches derived from constructing epigenetic hapfotype population structures.
  • standard genetic approaches including but not limited to: single- or multi-locus genetic mapping; quantitative trait locus (QTL) mapping; or association mapping approaches derived from constructing epigenetic hapfotype population structures.

Abstract

L'invention se rapporte au domaine du profilage de méthylation de l'ADN. L'invention porte, en particulier, sur un procédé qui fait appel à une conversion de la cytosine médiée par le bisulfite, suivie d'une amplification aléatoire au moyen d'un modèle d'amorce unique, et sur un procédé de traitement pour biopuces ou autres systèmes d'analyse de séquences à haut rendement. Le procédé de l'invention permet d'améliorer la sensibilité de la détection et d'augmenter la résolution par rapport à l'état actuel de la technique en matière de détection de la méthylation de l'ADN à l'échelle du génome.
PCT/EP2008/055377 2007-05-02 2008-04-30 Procédé d'amplification de l'adn WO2008135512A2 (fr)

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