WO2008135512A2 - Dna amplification method - Google Patents

Abstract

The present invention is in the area of DNA methylation profiling. In particular, the invention relates to a method of using bisulfite-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.

Description

DMA AMPLIFICATION METHOD

The present invention is in the area of DNA methylation profiling. In particular, 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,

Epigenetics modifications, heritable changes in gene expression due to DNA modifications without altering DNA sequences ', have a crucial role in the control of gene activity and nuclear architecture. Well characterized epigenetic mechanisms include covalent DNA methyiation modifications, histone amino-tail modifications, and replacement of histone variants. 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,

The repressive rote of DNA methylation on transcription is well characterized, but recent data have raised new questions about the ro!e and complexities of DNA methyiation. The involvement of DMA methylation in transcriptional gene silencing (TGS) results from methylated DNA inhibiting normal interactions with the transcriptional machinery and by interacting with MBD proteins that recruit repressive histone modifications and chromatin remodelling factors ¹. One of the examples for the repressive role of 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.

Given cytosine methylation is a central mechanism of epigenetic gene regulation and aberrant DNA methylation patterns can be associated with cancer, epigenomic profiling methods are an area of great research interest, A growing number of methods are being developed to detect DNA methyiatiαn patterns. Reviews for additional methods that are not discussed here are incorporated as references herein ^{8l 9}. The main focus within the scope of the present invention is on three commonly used techniques: methylation sensitive restriction enzymes, chemicai treatment of DNA with sodium bisulfite, methylated DNA immunoprecipitation and IVSBD (methyl binding domain) protein affinity purification.

The use of methylation-sensitϊve restriction enzymes (MSREs) assays the methylation state within the recognition sites ¹⁰, This method can be applied to Southern blot hybridizations, restriction genomic landscape scanning, AIMS or methylation sensitive amplified fragment length polymorphisms ^{11 12 10}. Related efforts have coupled MSREs to microarray approaches ⁵ or used the 5mC-dependant homing restriction nuclease McrBC ^{13 14}. Although these techniques can identify methylation polymorphisms, one disadvantage can occur due to the limited representation of the recognition motif across the genome. Additionally, not all methylation targets sites can be assayed with known MSREs, Further, some MSREs lack corresponding isoschizomers. More importantly, for MSRE approaches coupled to microarray platforms, the analytical resolution becomes a function of the restriction site specificity and the fractionation steps that may decrease the detection sensitivity. This has partly been overcome by the using a cocktail of 5 MSREs to identify roughly 1000 methylation polymorphisms on the active X chromosome ². However, the results demonstrate the limited information gained from MSRE-based techniques, even when using high density oligonucleotide arrays. Conversely, chemical treatment of DNA with sodium bisulfite allows the ability to detect methylation polymorphisms at each cytosine, but the treatment can be a difficult process. 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, bisulfϊte-mediated cytosine conversion identifies C-to-T transitions, analogous to single nucleotide polymorphisms (SNPs), to allow detection of methylation polymorphisms. Although PCR- based cloning at locus-specific sites for sequencing provides methylation detection at the base pair level; the effort is laborious, requires locus-specific primers, and sequencing multiple clonal reactions. Further, this method has not previously been applied to genome- wide analyses. Recently, a high-throughput sequencing effort directly sequenced PCR products along a tiling path methyiation profile on human chromosome 6, 21 , and 22 ^1S, but this requires significant sequencing capabilities and appears difficult to apply for genome- wide analysis. To expedite methylation detection, several related techniques avoid cloning and sequencing ^{19 20 21 22 23 24} provided a priori methylation targets are available and suitable primers can be designed for efficient assays. Yet, the analytical specificity of these approaches is limited to methylation changes only within the primer annealing sites²⁴, the RE site ²¹ or probe design for single base extension assays . Although several reports for microarray-based detection have been published using either MSREs or bisulfite conversion, these analyses were focused on a relatively small number of known, locus- specific targets ^{2S 26} and has not been reported for genome-wide de novo detection of methylation polymorphisms.

Although bisulfite analysis can quantify methylation levels at the base pair level, the treatment inherently possesses several disadvantages. One problem is depurination- induced fragmentation resulting from the bisulfite treatment. Another concern is that residua! bisulfite salts can co-precipitate with the converted DNA and cause inhibitory effects towards downstream applications. Efficient primer design for PCR amplification can be problematic. Also, the cloning steps required for sequencing are laborious and time consuming. Furthermore, beyond the above difficulties associated with conventional bisulfite sequencing efforts, the primary limitation towards applying btsulfite-mediated cytosine conversion to genome-wide analyses has been due to the difficulty in reproducibiy amplifying treated DNA without amplification bias.

As a result, recent efforts have focused on enrichment strategies using methyl binding domain proteins coupled with affinity chromatography or anti-5mC antibodies to immunoprecipitate methylated DMA ^{27 28 3 4}, This could lead to direct sequencing efforts or can be coupled to oligonucleotide arrays for genome-wide methylatioπ profiling ^{3 4}. Although the method is suitable for enriching methylated DNA, a disadvantage results from relatively large fragment following sonication or nebulization treatment ²⁹. if a DNA fragment contains sufficient methylation for enrichment using the above techniques, but also co-precipitates adjacent, unmethyiated DNA, faise positive signals can occur while simultaneously decreasing resolution and sensitivity. Last, although immunoprecipitating methylated DNA is ideal for methylated CpG islands with greater than 2% methylation, it is not clear how effective this method fs at detecting methylated non-CpG islands or methylated non-CpG sequences ²⁷.

There was therefore a need for a method for amplification of sodium bisulfite-treated DNA for genome-wide DNA methylation profiling, which is capable of overcoming the limitations inherent to the bisulfite treatment by developing a non-biased amplification method that is reproducible for genome-wide DNA methylation profiting on high density oligonucleotide arrays.

This need is met by providing the method according to the invention and as described herein below, which allows improved detection sensitivity and high resolution over the current state of the art methods of genome-wide detection of DNA methylation.

In one embodiment, 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. in 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..

In one embodiment of the invention, the polynucleotide sample is a DNA sample. In one embodiment of the invention, the polynucleotide sample is a RNA sample.

In one embodiment of the invention, the polynucleotide is from an animal, particularly a mammal, but especially a human.

In one embodiment of the invention, the polynucleotide is from a plant, particularly a plant of agronomic importance such as a field crop or a vegetable plant.

In a specific embodiment of the invention, the genomic DNA is digested for sample preparation using restriction enzymes, particularly enzymes lacking cytosine residues within the recognition site.

In another specific embodiment of the invention, 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.

In still another specific embodiment of the invention, 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. It is within the spirit of the current invention that an alternative treatment of bisuifite-treated DNA with uracil DNA glycosylase (UDG) prior to random amplification may decrease the relative proportion of converted DNA templates, thus increasing the fluorescent signal intensities differences between sampies to better facilitate the detection of methylatϊon polymorphisms, in still another specific embodiment of the invention, the sodium bisulfite and sodium hydroxide molecules are efficiently removed by the end of the treatment.

In one embodiment of the invention 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. Thus, the 3' end of the primer wilf be complementary to random sites throughout the target DNA segments. Sn a specific embodiment, 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;

(ii) 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;

(iiϊ) a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, wherein 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; (iv) 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.

In a specific embodiment of the invention, 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-pπmer 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 5¹ 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.

In another specific embodiment, 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

(v) the primary reaction product; (vi) 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 3¹ end;

(vii) a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, dUTP wherein G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidyiic nucleotides, C cytidylic nucleotides and U uracyiic nucleotides, which nucleotides may be labeled, particularly with a fluorescence marker and wherein 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 especially 4:1; (viii) 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.

in still another specific embodiment, the invention 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

(ix) the primary reaction product; (x) 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 3¹ end; (xi) 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

Taq DNA polymerase.

In particular, 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.

In one embodiment, 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 more particuiarly of at least 15 defined, but especially of at Ieast 20 defined nucleotides and up to at Ieast 30 - 40 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 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.

In one embodiment of the invention, the polynucleotide sample is a DNA sample. In one embodiment of the invention, the polynucleotide sample is a RNA sample.

In one embodiment of the invention, the polynucleotide is from an animal, particularly a mammal, but especially a human.

In one embodiment of the invention, the polynucleotide is from a plant, particularly a plant of agronomic importance such as a field crop or a vegetable plant.

In a specific embodiment, 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);

(U) 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 5¹ of the random nucleotides; (Hi) a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, wherein G is understood to represent guanyiic nucleotides, A adenylic nucleotides, T thymidyfic nucleotides and C cytidylic nucleotides, (iv) a DNA polymerase, particularly a heat sensitive DNA polymerase exhibiting primer displacement activity but essentially no exonuciease activity, more particularly a T7 DNA polymerase, but especially a modified T7 DNA polymerase such as, for example, a Sequenase possessing primer displacement activity that results in longer polymerization products.; b. optionally, 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.

In another specific embodiment, 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:

(i) the primary reaction product; (ii) 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 3¹ end; (iii) a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, dUTP, wherein G is understood to represent guanyiic nucleotides, A adenylic nucleotides, T thymidylic nucleotides, C cytidylic nucleotides and U uracylic nucleotides, wherein 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 is especially 4:1 ; (iv) a DNA polymerase, particularly a heat-stable DNA polymerase, more particularly a Taq polymerase.

In another specific embodiment, 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 π 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^! end of the first primer but Sacking the random sequence of nucleotides at its 3' end; a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, wherein G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidylic nucleotides, and C cytidylic nucleotides; (vii) a heat-stable DNA polymerase, particularly a heat-stable DNA polymerase, more particularly a Taq polymerase

In one aspect of the invention, 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 iπ- 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.

In one embodiment of the invention, the amplified polynucleotide can be applied towards conventional, locus-specific PCR-based cloning. Following sodium bisulfite treatment of a polynucleotide sample, particularly a DNA sample, 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. Alternatively, the amplified polynucleotide, particularly the amplified DNA, can be analyzed using standard cloning techniques known to those skilled in the art. 32

In one embodiment of the invention, the amplified polynucleotide can be applied towards quantitative PCR. For example, 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.

Alternatively, 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. On one end of the hybridization probe (either the 5¹ or 3' end), there is a donor fiuorophore, and on the other end, an acceptor moiety. When used in PCR, 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} . Hence, the fluorescence signal intensity may be used to quantitatively measure the abundance of methylation polymorphisms.

Similarly, 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.

Further, amplified DNA obtainable in a process according to the present invention may be used for the 5' nuclease PCR assay, referred to as a "Taqfvlaπ" assay ³¹ according to the previously described method.

in one embodiment of the invention, 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 pyrosequβncing, massively parallel signature sequencing (MPSS), sequencing by synthesis (SBS)₁ or clonal single molecule array (CSMA). Further, 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 process according to the present invention may also be applied to such sequencing techniques. Alternatively, enrichment of methylated DNA could be obtained by digesting DNA with methylatioπ-sensitive restriction enzymes followed by gel purification to obtain specific DNA fractions to be bisuifite-converted, amplified, and sequenced, as described above,

In one embodiment of the invention, 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.

in one embodiment of the invention, 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.

In one embodiment of the invention, 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.

Likewise, 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.

!n one embodiment of the invention, 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.

in one embodiment of the invention, 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.

In one embodiment of the invention, 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) aϋotetraploidy, and (c) aneuploidy to determine the association between the DNA methylation state and the organism's ploidy level.

In one embodiment of the invention, 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.

In one embodiment of the invention, 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. In one embodiment of the invention, 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.

in one embodiment of the invention, 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.

in one embodiment of the invention, 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: uπdesired 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.

Brief Description of the Figures

Figure 1: Overview of the amplification method Overview of the Bisulfite Methylation Profiling (BiMP) method. Graphical representation of a DNA template with variable DNA methylation levels (purple, low DNA methyiation; blue, moderate DNA methylation; green, high DNA methylation) and the expected degree of sequence conversion resulting from sodium bisulfite treatment, indicated below the DNA, respectively. Following bisulfite treatment, standard "state of the art" methods render unreliable resufts (data not published). Here, using the BiMP method, bisuifite-converted DNA is amplified and labeled for array hybridization experiments, with probe cotors corresponding to the methyiation levefs of the originai DNA template. 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.

Figure 2: Graphical representation of the Bisulfite Methyiation Profiling (BiMP) amplification method

Following bisulfite treatment, converted, single-stranded DNA molecules are mixed with the oligonucleotide primer comprised of two portions: a 5¹ region comprised of a defined nucleotide sequence and the 3' region comprised of a random nucleotide sequence, such as 4 random nucleotides. In the first step, 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). In step two, the newly synthesized double-stranded DMA templates are amplified using PCR in the presence of dNTP, including dUTP to facilitate fragmentation. In the first PCR cycle, the known primer will anneal at complementary sites (a) and initiate template elongation (b). in subsequent PCR cycles, the amplification process proceeds using the known primer sequence (c). The present invention provides a genome-wide method of DNA methylation profiling independent from the limitations associated with current methods applied for this purpose. In one embodiment of the invention a cytosine conversion method, particularly a bisulfite- mediated 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 ),

In particular, 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. in 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,

fn a first step, 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. Additionally, in a further preferred embodiment of the invention, the fragmentation caused by depurination is limited using known methods. Within the scope of the present invention 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). In particular, 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. 1A) and the presence of the multi- copy 180bp pericentromeric repeat signal observed in the dot blot hybridization (see Reinders et a/, Fig. 1B). 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.

In particular, 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 5¹ 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).

In a specific embodiment, 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. After the heat denaturing, the sample is cooled, particularly to about 4 to 10⁰C 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. After the primer C annealing step, while the temperature is cooled , 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. After adding the polymerase reaction mix to the DNA reaction mixture, 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. For the second, repeated polymerase reaction, 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.

Because of the second cycle, 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 3¹ 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 diiutioπ factors may be acceptable with a particular set of reactions and is encompassed by the present invention.

These primary reactions are followed by a second amplification round, wherein the first round primary amplification product is amplified by means of a second oligonucleotide primer comprising essentially only the second defined portion of the first oligonucleotide primer used in the primary reactions. In particular, 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.

In a specific embodiment of the invention, 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. In one embodiment of the invention, 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 94⁰C 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 94⁰C, a

30 second annealing step at about 40⁰C, a 30 second annealing step at about 50⁰C, a 1 minute DNA polymerase extension step at about 72⁰C. This cycle is repeated from 20 to about 35 times, particularly from 25 to 30 times, but especially 30 times. The reaction is then held at about 72⁰C, from 5 - 10 minutes, preferably 10 minutes to complete polymerizations. The sample is then cooled to 4 to 10⁰C. It is understood that varying buffer compositions, concentration of certain reagents, and variant DNA polymerases for

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 endoπucJease 1 (APE 1) mediated fragmentation. In a specific embodiment of the invention, 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. In particular, 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^CC, followed by about 2 minutes incubation above 92⁰C₁ 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.

After suitable probe fragmentation of the bisulfite-treated amplified DNA, 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.

In a specific embodiment of the invention, fragmented DNA is labeled in a TdT buffered reaction using terminal deoxynucleotidy! transferase (TdT) in the presence of a biotiπylated compound. Commercial labeling 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^πC, heated for about 10 minutes at about 70⁰C 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, DMSO₁ B2 control oligo

(3πm), RNA hybridization spikes, and water. The hybridization mixture is then heated to about 94⁰C 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 μg of labeled, fragmented DNA was hybridized for 16hours at 45°C.

Following hybridization, 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

To those skilled in the art, hybridizing labeled DNA probe samples to a microarray is an ideal method to simultaneously coilect information about a large number of DNA sequences. Here, 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. Typically, a sample of fiuorescentiy fabeied nucleic acids (probe) is hybridized to the microarray. Subsequently, the arrays are washed and scanned to obtain the fluorescent signal intensity per feature is detected and the biological information is inferred from the emitted signal per feature. 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. Within the scope of the present invention, 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

20% Bieach, then water, then ethanol. Use steriie, filtered tips throughout the procedure to avoid cross sample contamination.

2. Set a water bath or heat block to 65°C.

3. Record date, plant number, and fresh weight in lab journai, a. Entry 1 is CoI-O, the wild type Arabidopsis thaliana reference accession. Entry

2 is met1~3, a null mutant allele of the Arabidopsis DNA methyltransferasel gene (AT5G49160) as previously characterized ^3e. b. Prepare one tube per replicate per entry with giass beads and label accordingly. c. Add tissue from 6 or more plants to the tubes and freeze in liquid nitrogen. d. Grind for 10 seconds and return to liquid nitrogen, avoid thawing. e. Repeat grinding step.

4. Add beta-mercaptoethanol (Sigma) to CTAB buffer (20 μi per 10 mi solution) just prior to use. Add 1 ,0 mi of 2X CTAB buffer per tube. Vortex until tissue is suspended into solution.

5. Incubate 0.5 to 3 hours at 65°C. After 15 minutes, add 1/100 volume N~sodium lauroylsarcosine salt solution (Fluka), mix and return to 65⁰C incubation.

6. Centrifuge 10^! at high speed at room temperature.

7. Transfer supernatant to a 2.0ml tube. Add equal voiume of Phenol (Sigma). Mix vigourousiy for 5 - 30'.

8. Centrifuge 5' at 1600Og at room temperature.

9. Transfer aqueous phase to a new 2.0mi tube. Add equal volume of phenol:chloroforrn:isoamyl aicohoi (25:24:1) (Sigma). Mix vigourousiy for 5 - 30'.

10. Centrifuge 5' at 1600Og at room temperature (optional step: 3^rd Chioroform extraction step)

11. Transfer aqueous phase to a new tube. Add 0.7X voiume isopropanoi, incubate 20' at -80⁰C, and spin 15' at 1600Og at 0⁰C.

12. Remove supernatant, but keep on hold. 13. Resuspend pellet with 0.2 ml TE buffer.

14. Add 1μg Rnase A and incubate for 30' at 50⁰C. Add Proteinase K (Roche) to a final concentration of 1.0 μg/m! and incubate 0.5-1 δhrs at 50^pC.

15. Add equal volume of phenol:chioroform:isoamyl alcohol (25:24:1). Mix 5 - 10'. 16. Centrifuge 5' at 1600Og at room temperature. Transfer the aqueous phase to a new, steriie tube.

17. Add equal volume of chloroform: isoamy! alcohol (24:1 ). Mix for 10'.

18. Centrifuge 10' at 1600Og at RT.

19. Transfer the aqueous phase to a new, sterile tube. Ethano! precipitate with 1/10 volume 3.0M Sodium acetate (pH 5.2) and 2.5X volume ethano!.

20. Wash the pellet twice with 70% ethanol wash. Remove all residual ethanol. Do not air dry for ionger than 15 minutes

21. Resuspend in 30-1 OOμL Tris/EDTA buffer.

22. Assess the sample quality: a. Quantify the DNA concentration using a fϊuorometer. b. Assess the DNA quality and integrity using a 1 % agarose gel stained with ethidium bromide. c. Assess the DNA purity using the 260/280 ratio observed in a spectrophotometer,

Example 2: Sample Preparation and Bisulfite Treatment

1. Digest a 4.0μg genomic DNA sample with a 2- to 10-fold excess of Dra! restriction enzyme (Promega), as recommended by the manufacturer.

2. Adjust the volume of the reaction to 100μL with water or TE buffer. 3. Transfer supernatant to a 2.0ml tube. Add equal volume of phenol (Sigma). Mix for

5-10'.

4. Centrifuge 5' at 1600Og at RT.

5. Transfer aqueous phase to a new 2.0m! tube. Add equal volume of pheno!:chloroforrn:isoamy! alcohol (25:24:1) (Sigma). Mix for 5-10'. 6. Centrifuge 5' at 1600Og at RT.

7. Transfer aqueous phase to a new tube.

8. Add equat volume of Chioroform. Mix for 5 - 10'. 9. Centrifuge 5' at 1800Og at RT.

10. Transfer aqueous phase to a new tube. Add 2.0X volumes of ethano! or 0.7X volume of isopropanol, incubate 20^! at -80⁰C or 16 hours overnight and spin 15' at 1600Og at 0⁰C for 30 minutes. 11. Remove supernatant, but keep on hold,

12. Wash the peliet twice with 70% ethanol. Remove ail residual ethanol. Do not air dry for longer than 15 minutes. 13. Resuspend the pellet in 23uL of RNase-free, sterile distilled water.

14. Quantify the DNA concentration using a fluorometer. Assess the DNA digestion using a 1% agarose gei stained with ethidium bromide. Assess the DNA purity using the 260/280 ratio observed in a spectrophotometer.

15. Adjust the sample volume to 100ng/μl and transfer a 20μL voiume to a sterile 200μL PCR reaction tube accordingly labeled.

16. For the bisulfite treatment, the EpiTect Bisulfite modification kit (Qiagen) has provided adequate sample conversion. Follow the manufacturers protocol as directed.

17. Take an aliquot of the sample representing 100 to 250ng of bisulfite converted DNA and ethanol precipitate by mixing 1/10 voiume 3.0 iVI Sodium acetate (pH 5.2} and 2.0X volumes of 100% ethanol, mix gently, and incubate at -20⁰C for 16 hours. 18. Precipitate the DNA by centrifuging the sample at 4⁰C for 30 minutes and maximum speed.

19. Wash the pellet twice with 70% ethanol. Remove all residual ethanol. Do not air dry for longer than 15 minutes.

20. Resuspend the peliet in 7μL of RNase-free, sterile distilled water.

Example 3: DNA Ampiification

1. Add 2.0μL of 5X sequenase buffer (USB) and 1.0μl_ of Primer C (40μM) (GTTTCCCAGTCACGATCNNNN) to each DNA sample. Additionally, prepare a no template control reaction and a positive non-treated bisulfite treated genomic DNA control.

2. Prepare the reaction mix comprised of : a. 1.OμL of 5X sequenase buffer (USB) b. 1.5μL of a dNTP mixture (1GmM) i. ClATP (IOmM) ii. dCTP (I OmM) iii. dGTP (10mM) iv. dTTP (10mM) c. 0.75μL DTT (0.1 M) d. 1.5μL of bovine serum albumin (500μg/ml) e. 0.3μL of Sequenase (13U/μL)

3. Place the DNA sample into a thermal cyder and perform the first cycle of random DNA amplification : a. Denaturation at 94.0⁰C for 2 minutes. b. Coo! to 10⁰C and hold for 5 minutes. During this step, open the reactions and add 5.0μL of the reaction mix to each sample, mix gently, and close. !f possible, pause the machine while performing this step. If the samples are removed, place on ice while adding the reaction mix. Avoid any contamination of the reactions. c. Allow random primer annealing as the temperature increase to 37^DC at a rate of 0.05⁰C s^"1. d. Allow 8 minutes incubation at 37°C for polymerase extension. 4. Perform the second cycle of random DNA amplification : a. Per sample prepare a sequenase dilution reaction mix comprised of: i. O.QμL of sequenase dilution buffer ii. 0.3μL of sequenase. b. Denaturation at 94.O⁰C for 2 minutes. c. Cool to 10⁰C and hoid for 5 minutes. During this step, open the reactions and add 1.2μL of the sequenase dilution reaction mix to each sample, mix gently, and close. If possible, pause the machine while performing this step. If the samples are removed, place on ice while adding the reaction mix. Avoid any contamination of the reactions. d. Allow random primer anneafing as the temperature increase to 37^DC at a rate of 0.05⁰C s^"1. e. Allow 8 minutes incubation at 37°C for polymerase extension. 5. Adjust the first round reaction volume to 60μL with RNase-free, sterile distilled water.

6. Set up three second round amplification reactions per sample. Additionally, prepare an additional no template control for this amplification step. Per sample, prepare the following reaction mix: a. 10.0μL First round reaction product b. 8.0μL of 25mM MgC!2 c. 20.0μL of 5X Promega GoTaq PCR amplification buffer d. 5.0μL of a dNTP mixture with i. dATP (10 mM) ii. dCTP (10 mM) ϋi. dGTP (IG mM) iv. dTTP (β mM) v. dUTP (2 mM) e. 1.OμL Primer B (100 pmo!/ui) (GTTTCCCAGTCACGATC) f. 1.0μL Taq DNA polymerase (Promega 10 U/ui) g. 65.0μL RNase-free, sterile distiiled water

7. Amplify the DNA using the following program: a. 94.0⁰C for 3.0 minutes b. 94.0⁰C for 30 seconds c. 40.0⁰C for 30 seconds d. 50.0⁰C for 30 seconds e. 72.0⁰C for 1.0 minutes f. Go to step b), and repeat 29 times g. 72.0°C for 10.0 minutes h. Hold at 4.0⁰C

8. Purify the amplified reactions using the PCR purification kit, such as the GenElute PCR clean-up kit (Sigma).

9. Combine the replicate reaction products into one sample and assess the sample quality: a. Quantify the DNA concentration using a fiuorometer. b. Assess the DNA quality and integrity using a 1 % agarose gel stained with ethidium bromide. c. Assess the DNA purity using the 260/280 ratio observed in a spectrophotometer. Use only samples with ratio values equal to or greater than 1.80. d. Ensure no background amplification has occurred with the negative controls. 10. Prepare a 9.0μg sample aliquot and ethanoi precipitate as previously described. 2X

70% ethano! wash. Remove all residual ethanoi- Do not air dry for longer than 15 minutes 11. Resuspend the pellet in 39.5μL of nuclease free sterile water. Make sure the peilet is completely resuspended. if necessary, vortex briefly and incubate the sample at 37⁰C for 20 minutes, or mix with a sterile, filtered pipet tip to resuspend. Store at 4°C

Example 4: DNA Fragmentation, Labeling, Hybridization

1. Prepare the fragmentation mix comprised: a. 39.5μL of amplified DNA (228.1 ng/μL) b. 4.8μL of 10X cDNA fragmentation buffer (Affymetrix) c. 1.5μL of UDG (10 U/μL) (Affymetrix) d. 2.25μL of APE 1 (100U/μL) (Affymetrix).

2. Mix the fragmentation reactions and briefly centrifuge the samples.

3. Incubate the reactions at: a. 37°C for 2 hours b, 93°C for 2 minutes c. 4°C for 2 or more minutes.

4. Mix the samples and briefly centrifuge, transfer 45μL of the sample to a new, sterile PCR reaction tube.

5. Analyze 1.0μL of the remnant sample for fragmentation anaiyis using the Agilent Bioanalyzed 2100 with the RNA 600 LabChip kit as directed by the manufacturer. 6. Prepare the DNA labeling mix comprised of: a. 12.0μL of 5X terminal deoxynucleotidyl transferase (TdT) buffer (Affymetrix) b. 2.0μL of terminal deoxynucleotidyl transferase (Affymetrix) c, 1.OμL DNA iabeiing reagent (5mM) (Affymetrix).

7. Add 15.0μL of the DNA labeling mix to the DNA sample, mix gently, and briefly spin,

8. incubate the reactions at: a. 37⁰C for 60 minutes b. 70⁰C for 10 minutes c. 4°C for 2 or more minutes.

9. Prepare the hybridization reactions by mixing: a. δQμL of the fragmented, labeled DNA b, 4.0μL of 3 nM B2 control oligonucleotides (Affymetrix) c. 12.5μL of 2OX RNA hybridization spike controls d. 120μL of 2X hybridization buffer e. 16.8μL of DMSO f. 36.70μL of sterile, nuclease free water.

10. Gently mix and briefly spin. 11. Heat denature at 99⁰C for 5 minutes,

12. Incubate at 45⁰C for 5 minutes,

13, Centrifuge at high speed in a centrifuge for 5 minutes.

14, Transfer 200μL of the hybridization cocktail to the pre-hybridized high density tiling array, as recommended by the manufacturer. 15. Hybridize the samples to the array at 45°C for 16 hours in a hybridization oven.

Example 5: Array Processing

Note: ali steps are followed as recommended by the manufacturer {Affymetrix). Briefly,

1. Enter the experiment information into the GeneChip Operating Software 2. Prepare the fluidics station

3. Prime the fluidics station

4. Wash and stain the arrays

5. Scan the arrays

6. Move the resulting data files to a working directory. Example 6: Data Analysis

1. Open the Affymetrix Tiling Analysis Software (TAS).

2. Define the analysis parameters a. Set the data paths to the working directory b. Set the export paths to the desired directory c. Set the normalization parameters i. Select scale intensity to 100 d. Scale i. Signal scale to Iog2 ii. P value scale to -1 Olog 10 e. Probe Analysis

L Bandwidth to 80 ii. Test type to one sided upper iii. Intensities to PM/MM f. Interval Analysis i. Threshold to 1-e005 ii. Less than threshold iii. Maximum Gap to 80 iv. Minimum run to 40

Upon bisulfite conversion, the expected result is an increased proportion of probes originating from uπmethylated DNA with lower fluorescent signal intensity in comparison to non treated DNA signal (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). Within the first histogram Interval, representing features with the lowest signal, approximately 1.2% and 0.004% of the bisulfite-treated and non-treated DNA probes, respectively, were detected indicating only minimal bias introduced from the amplification method. The expected increase of number of probes with a lower signal was observed in histogram bins 2 to 5, representing 92% and 95% of the Co! and met 1-3 datasets, respectively, in comparison to 42% of non-treated DNA reference. Using the tiling analysis software (Affymelrϊx), correlation coefficients for the biological replicates were calculated (see Reinders et a/, Supp Table. 1 ) and were similar to previously reports. Last, the technical reproducibility was assayed by performing hybridizations amplified from the independently ampiified technical replicates but using the same bisulfite-treated DNA. The 5 bϊsulfite-treated CoI and met1-3 technical replicates had correlation coefficients of 0.98 and 0.90, respectively (see Reinders et a/, Fig, 2).

Next, the biological reproducibility was assessed by comparing three hybridization datasets for Co!^8S+ and met1~3^BS÷, where plant material, DNA isolation, bisulfite treatment, "fourN"

IO amplification, labeling and hybridizations were ail performed independently. The correlation coefficients were 0.96 and 0.92 for the Coi^BS+ and met1-3^BS+ datasets, respectively (see Reinders et al, Supp Table 1). These results were compared to the mCIP DNA methylation profiling performed on the same Affymetrix platform for the same Arabidopsis strains ³ (data sets available at http://www.ncbi.nlm.nih.goy/prgjects/geQ/, accession number

!5 GSE5^Q94). The calculated mCIP correlation coefficients were 0.94 and 0.92 for the wild type and meti-3, respectively (see Reinders et al, Supp Table 1), and thus, were similar to the BiMP values. C hro mo so me- wide bisulfite- mediated methylation profiles were generally consistent with the expected results. First, the hybridization results clearly demonstrated a predominant loss in signal intensity for bisulfite-treated samples, with a proportion of 0 features retained the signal intensity (see Reinders et al, Fig. 3 and Supp Fig. 3). Specifically, increased methylation levels were observed at the heterochromatic regions compared to euchromatic regions and heterochromatic features, such as the "knob" on chromosome 4S were visible (see Reinders et al, Fig. 3). 5 Given the reproducibility of the BiMP datasets and the methyiation analyses at heterochromatic regions, single-copy loci were examined known to change methylation levels in met1-3 mutants. For example, the FWA and SUPERMAN (SUP) genes are hypomethylated and hypermethyiated, respectively, in met1-3 relative to the wild type ³². BiMP analysis of the diagnostic tandem repeats within the 5¹ region of FWA clearly showed0 the loss of methylation in met1~3 (see Reinders et al, Fig. 4A). Conversely, the 5' region of the SUP locus revealed hyperrnethylation in met1~3 (see Reinders et al, Fig. 4B). When mCiP and BiMP feature-ievel signal intensities were each processed using Affymetrix's Jjiing Analysis Software (TAS), the improved sensitivity and resolution of BiMP over mCIP was confirmed (see Reinders et al, Fig. 3B and 4). Previously, the mCIP results (available at http://epigenomics.mcdb.ucia.edu/DNA.meih/ and http://signal.satk.edu/cqi- famMethvtorne) were analyzed using a two-state hidden Markov model (HMM) based on probe-level t statistics ³. However, this approach is likeiy to miss short intervals as differentiaily methylated, for example at the SUP locus (see Reinders et al, Supp Fig. 4). This is likeiy due to the observed high rnCIP signal intensity in Columbia across the 5' genie region which could mask adjacent iocaf changes (see Reinders et at, Fig. 4B) or because the levels were classified as indifferent from the average global distribution.

Since the 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. Considering that total wild-type levels of ^mC ranged from 4-6% ³³, the observed changes are rather drastic. On average, the regions that gained or lost methyiation were 121 and 101bp in length, respectively, indicating that methyiation changes predominantly occur within relatively short intervals that would be difficult to visualize with mCIP given its maximum resolution estimated at 400bp ³. These hyper- and hypomethylated intervals were analyzed for the relative proportions of sequence motifs containing cytosines. Notably, asymmetric CHH sites were more frequent in the hypermethyiated compared to the hypomethylated intervals (see Reinders et al, Supp Table 2 and 3).

To validate novel methyiation polymorphisms revealed by BiMP conventional bisulfite sequencing was used. Increased methyiation in met 1-3 was observed at the REPRESSOR OF SILENCING 1 (ROS1) and APETALA 3 (AP3) genes (see Reinders et al, Fig. 5A, 5B and Supp Fig. 9) and the sequencing results supported the BiMP data (see Reinders ef al, Fig. 5C and 50), Importantly, methyiaiion in mei1-3 also included CG sites that were not methylated in the wild type (see Reiπders βt a/, Fig. 5D and Supp Fig. 10). At the assayed AP3 region, the signal difference was slightly below the chosen cutoff, suggesting that our analysis was a conservative estimate of differentially methylated regions. Methylation profiles between mCIP and BiIvIP at additional ioci were identified (see Reinders et al, Supp Fig. 11) and examined by bisulfite sequencing. The BiMP results were supported (see Reiπders et at, Supp Fig. 12).

in summary, 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 epϊgenomic 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.

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Claims

1. A method for detecting methySation patterns 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. in 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 5¹ end comprises a defined nucleotide sequence, particularly a sequence of at feast 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 cytosine methylation and methylated CpG islands in said polynucleotide sample.

2. A method according to claim 1 , wherein the random nucleotide sequence comprises between 3 and 5 random nucleotides.

3. A method according to claim 1, wherein the random nucleotide sequence comprises 4 random nucleotides.

4. A method according to any of the preceding claims, wherein said defined nucleotide sequence comprises at ieast 15 defined nucleotides.

5. A method according to any of the preceding claims, wherein said defined nucleotide sequence comprises at least 20 defined nucleotides.

6. A method according to any of the preceding ciaims, wherein the polynucleotide sample is a DNA sample, particularly a genomic DNA sample,

7. A method according to any of the preceding claims, wherein polynucleotide sample is a RNA sample.

8. A method according to claim 6 wherein the genomic DNA is purified for sample preparation by removing RNAs and proteins to obtain pure DNA without detectable RNA or protein contamination.

9. A method according to any one of claims 6 or 8, wherein the genomic DNA is digested for sample preparation using restriction enzymes, particularly enzymes lacking cytosine residues within the recognition site,

10. A method according to claim 9, wherein the digestion products are 30KB or iess.

11. A method according to claim 9, wherein the digestion products are in a range of between 0.5-10Kb.

12. A method according to claim 9, wherein the digested DNA is purified from the restriction enzymes.

13. A method according to any of the preceding claims, wherein the digested DNA is treated with sodium bisulfite such as to convert essentially all non-methylated cytosines to uracil, and to maintain ail methylated cytosines as cytosines; and to minimize fragmentation effects due to depurination during the treatment.

14. A method according to claim 13, wherein bisuifite-treated DNA is treated with uracil DNA glycosylase (UDG) prior to random amplification.

15. A method according to claim 13, wherein adaptor motecuies are ϊigated to the DNA to incorporate a known primer sequence before, during or after, the bisulfite conversion, or the amplification process.

18, A method according to any of the preceding claims, wherein the sodium bisulfite and sodium hydroxide molecules are efficiently removed by the end of the treatment.

17. A method according to any of the preceding claims, wherein in first round amplification a primary reaction mixture is provided comprising:

(i) the converted polynucleotide sample produced in step a); (ii) 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* of the random nucleotides;

(iii) a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP_r dGTP, dTTP, wherein G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thymidylic nucleotides and C cytidylic nucleotides, (iv) a DNA polymerase, particularly a heat sensitive DMA polymerase exhibiting primer displacement activity but essentially no exonuclease activity.

18. A method according to any of the preceding claims, wherein the first round amplification is repeated at least once comprising denaturing the primary amplification product and repeating step b) in a second amplification cycie allowing the random segments of the first oligonucleotide primer to anneal to their complementary sequences,

19. A method according to any of the preceding claims, wherein said polymerase used in first round amplification is a T7 DNA polymerase, but especially a modified T7 DNA polymerase such as, for example, a Sequenase possessing primer displacement activity that results in longer polymerization products.

20, A method of any of the preceding claims, wherein in second round amplification a secondary reaction mixture is provided comprising (i) the primary reaction product; (ii) 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' end; (Hi) a mixture of nucleotides (d NTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, dUTP, wherein G is understood to represent guanyfic nucleotides, A adenylic nucleotides, T thyrnidylic nucleotides, C cytidylic nucleotides and U uracylic nucleotides; (iv) a DNA polymerase, particularly a heat stable DNA polymerase,

21. A method of any of the preceding claims, wherein in second round ampiification a secondary reaction mixture is provided comprising

(i) the primary reaction product;

(ii) 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' end; (iii)a mixture of nucleotides (dNTPs), particularly a mixture of dATP, dCTP, dGTP, dTTP, wherein G is understood to represent guanylic nucleotides, A adenylic nucleotides, T thyrnidylic nucleotides, and C cytidylic nucleotides; (iv)a heat-stable DNA polymerase, particularly a heat-stabie DNA polymerase,

22. A method according to claim 18, wherein 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 especially

4:1 ,

23. A method according to any one of claims 18-20, wherein said heat-stabie polymerase is a Taq DNA polymerase.

24.A method of any of the preceding claims, wherein the dNTPs are labeled with a detectable probe molecule to facilitate detection.

25.A method according to claim 22, wherein the dNTPs are labeled with a fluorescence marker, particularly a biotin.

26. The method of according to any of the preceding claims, wherein amplified DNA at a concentration equal to or greater than 225ng/μL is fragmented using: (a) equal to or greater than a final concentration of 10U/μL UDG; (b) equal to or greater than a final concentration of 100 U/μL of APE-1 ; (c) incubated at conditions of equal to or greater than 37⁰C for equal to or greater than 60 minutes.

27.A method according to any of the preceding claims, wherein methyiation polymorphisms are identified by hybridizing the amplified DNA to an array, particularly (a) a low density macroarray; (b) a spotted DNA micrσarray ; (c) a printed DNA microarray, (c) a mirror-based array design; or (d) an oligonucleotide array.

28.A method according to claim 25, wherein the amplified DNA is labeled with a detectable probe molecule.

29. A method according to ciaim 26, wherein the amplified DNA is labeled with a fluorescence marker, particularly a biotin.

30. A method according to any of the preceding claims, wherein methyiation polymorphisms are Identified using a method selected from the group consisting of (a) conventional, locus-specific PCR-based cloning; (b) quantitative PCR detection; (c) random "shotgun" genome sequencing approaches; (d) ultra high-throughput picoliter pyrosequencing techniques; (e) ultra high-throughput massively parailei signature sequencing {MPSS), sequencing by synthesis (SBS), or dona! single molecule array (CSMA); (f) "bead array" technologies using silica beads that self assemble in microwefls on a substrate(s), and (g) allelic discrimination assays using either single base extension of 5'exonuclease reporter probe ("Taqman") assays, including microfluidic "card" formats, barcode chips, and bioelectronic chips; (h) design of methylation-specific oligonucleotide arrays or microfluidic "card" formats derived from in-sifico conversion of sequences detected as DNA methyiation polymorphisms identified by hybridizaton of the amplified DNA to standard, commercially available oligonucleotide arrays or custom-designed "spotted" cDNA arrays.

31. A method according to any of the preceding claims comprising a validation step.

32.A method of determining the association between the DNA methyiation profile and the transcription profiie of a polynucleotide, comprising obtaining the methyiation profile of said polynucleotide by means of a method according to any one of claims 1-31 and comparing the DNA methyiation profile with the transcription abundance of said polynucleotide.

33. The method according to claim 32, wherein the transcription profile of a polynucleotide is detected with a microarray, ideally using identical platforms.

34. The method of determining the association between the DNA methyiation profile and the chromatin state profile of a polynucleotide, comprising obtaining the methyiation profile of said polynucleotide by means of a method according to any one of claims 1-29 and comparing the methyiation profile with the chromatin state of said polynucleotide including, but not limited to: (a) histone methyiation, (b) histone acetyiation, (c) histone phosphorylation, (d) histone ubquitination, (e) histone sumoylation, (f) histone citrullination, (g) histone ADP ribosyiation, (h) combinations or permutations of the above histone modifications and (i) where enriched nucleic acid fractions associated with the previously mentioned histone modifications are subsequently sodium bisulfite- treated for detection of methyiation polymorphisms.

35.A method of determining the association between the DNA methyiation state and chemical treatment comprising obtaining the methyiation profile of a polynucleotide by means of a method according to any one of claims 1-31 and comparing methyiation profiles of a polynucleotide sample following pharmacological treatments of a human or an animal with chemicals having the potential of affecting epigenetic states.

36. A method of claim 35, wherein the chemicais are selected from the group consisting of (a) azacytidine, and related derivatives such as 5-aza-deoxycytidine, or decitabine (DAC)₁ (b) TSA, and (c) other HDAC inhibitors.

37.A method of determining the association between the DNA methyiation state and copy number of a polynucleotide, comprising obtaining the methyiation profile of said polynucleotide by means of a method according to any one of claims 1-31 and comparing the methyiation profile of said polynucleotide with the copy number of said polynucleotide:

38.A method of determining the association between the DNA methyiation state and the organism's ploidy level, comprising obtaining the methyiation profile of a polynucleotide by means of a method according to any one of claims 1-31 and comparing the DNA methyiation profile of said polynucleotide in response to changes in the pioidy level of an organism.

39.A method according to claim 38, wherein a ploidy levei selected from the group consisting of (a) autotetraploidy, (b) aϋotetrapioidy, and (c) aπeuploidy is determined.

40. A method of comparing the methyiation profile within a human or an animal patient before and after having been subjected to stem ceil therapy by obtaining the respective methyiation profiles by means of a method according to any one of claims 1-31 and comparing the methyiation profile prior and after stem cell therapies.

41. A method of determining the association between the DNA methyiation state and RNAi treatment of a polynucleotide, comprising obtaining the methyiation profile of said polynucleotide by means of a method according to any one of claims 1-31 and comparing the DNA methyiation profile of said polynucleotide and comparing the methyiation profile prior and after RNAi treatment.

42.A method according to claim 39, wherein said RNAi treatment is selected from the group consisting of siRNA, miRNA, piRNA, rasiRNA, nat-siRNA, tran siRNA.

43. A method of determining the association between the DNA methyiation state and a plurality of tissues and development states, comprising obtaining the methyiation profile of different tissues, tumor samples, different development stages, or samples comprised of a single to multiple eel! levels by means of a method according to any one of claims 1-31 and comparing the methyiation distribution of said tissues, tumor samples, different development stages, or samples comprised of a single to multiple cell levels.

44.A method for ampiification of a genomic polynucleotide sample, particularly a DNA or a RNA sample, comprising a polynucleotide treated with a modifying agent that modifies unmeihyiated cytosine to produce a converted nucleic acid, particuiarly a sodium bisuifite-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. in 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, particularly a sequence of between 3 and 5 nucleotides, but especially a sequence of 4 nucleotides, and the second portion at its 5' end comprises a defined nucleotide sequence, particularly a sequence of at ieast 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).

45. Use of the amplified DNA obtainable in a process according to claims 44 as an input for hybridization to a set of probes of different nucieobase sequences, where (a) the non- hybridized probes are separated and the hybridized probes are analyzed in a mass spectrometer; (b) the peak pattern obtained from the mass spectra are assigned to the rnethylation pattern; and (c) the data obtained are compared with a database to lead to the detection of methylation polymorphisms.

46. Use of the amplified DNA obtainable in a process according to claim 44 for application towards the identification of markers for diagnostic or prognostic evaluation of samples for associations to disease conditions, notably cancer.

47. Use of the amplified DNA obtainable in a process according to claim 44 for determining whether a treatment or therapeutic agent, or combination thereof, will be effective in treating the condition or for selecting alternative treatments options.

48. Use of the amplified DNA obtainable in a process according to ciaim 44 for the identification of epigenetic modifications associated with epigenetic reprogramming, developmental differentiation, or differentiation.

49. Use of the amplified DNA obtainable in a process according to claim 44 in 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,

50. Use of the amplified DNA obtainable in a process according to claim 44 for comparing the DNA methyiation profile of the nucleic acid in response to changes in the ploidy level of an organism, including, but not limited to: (a) autotetraploidy, (b) aiiotetraploidy, and (c) aneuploidy to determine the association between the DNA methylation state and the organism's pbidy level.

51 , Use of the amplified DNA obtainable in a process according to claim 44 for 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 methyiation on the relative fitness of first generation progeny, e.g. hybrid vigor (heterosis), (b) subsequently self-fertilized 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 methyJation state and the organism's fitness level.

52. Use of the amplified DNA obtainable in a process according to claim 44 for comparing the methylation profile before, during or after stem cell therapy treatments for either diagnostic or prognostic purposes.

53. Use of the amplified DNA obtainable in a process according to claim 44 for comparing the methylation profile 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.

54. Use of the amplified DNA obtainable in a process according to claim 44 for comparing the methylation profile before, during or after gametogenesis of a specimen or patient for either diagnostic or prognostic purposes.

55. Use of the amplified DNA obtainable in a process according to claim 44 in the diagnosis and/or prognosis of adverse events in a patient, particularly of adverse events selected from the grop consisting of: undesired 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; cardiovascuiar disease, malfunction and damage; malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, 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.