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  Methods of analysis of methylationUSPTO Application #: 20080108073Title: Methods of analysis of methylation Abstract: Methods for determining the methylation status of a plurality of cytosines are disclosed. In some aspects genomic DNA target sequences containing CpGs are targeted for analysis by multiplex amplification using target specific probes that can be specifically degraded prior to amplification. The targets may be modified with bisulfite prior to amplification. In another aspect targets are cut with methylation sensitive or insensitive restriction enzymes and marked with a tag using the target specific probes. The presence or absence of methylation may be determined using methylation sensitive restriction enzyme or bisulfite treatment. Detection in many embodiments employs hybridization to tag arrays, genotyping arrays or resequencing arrays. (end of abstract) Agent: Affymetrix, Inc Attn: Chief Ip Counsel, Legal Dept. - Santa Clara, CA, US Inventors: Shivani Nautiyal, Malek Faham USPTO Applicaton #: 20080108073 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20080108073. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] The present application claims priority to U.S. application No. 60/862,735 filed Oct. 24, 2006, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0002] The genomes of higher eukaryotes contain the modified nucleoside 5-methyl cytosine (5-meC). This modification is usually found as part of the dinucleotide CpG. [0003] Cytosine is converted to 5-methylcytosine in a reaction that involves flipping a target cytosine out of an intact double helix and transfer of a methyl group from S-adenosylmethionine by a methyltransferase enzyme (Klimasauskas et al. Cell 76:357-369, 1994). This enzymatic conversion is the only epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic development (Bird, Cell 70:5-8, 1992; Laird and Jaenisch, Human Mol. Genet. 3:1487-1495, 1994; and Li et al. Cell 69:915-926, 1992). [0004] The frequency of the CpG dinucleotide in the human genome is only about 20% of the statistically expected frequency, possibly because of spontaneous deamination of 5-meC to T (Schoreret et al. Proc. Natl. Acad Sci. USA 89:957-961, 1992). There are about 28 million CpG doublets in a haploid copy of the human genome and it is estimated that about 70-80% of the cytosines at CpGs are methylated. Regions where CpG is present at levels that are approximately the expected frequency are referred to as "CpG islands" (Bird, A. P., Nature 321:209-213, 1986). These regions have been estimated to comprise about 1% of vertebrate genomes and account for about 15% of the total number of CpG dinucleotides. CpG islands are typically between 0.2 and 1 kb in length and are often located upstream of housekeeping and tissue-specific genes. CpG islands are often located upstream of transcribed regions, but may also extend into transcribed regions. About 2-4% of cytosines are methylated and probably the majority of cytosines that are 5' of Gs are methylated. Most of the randomly distributed CpGs are methylated, but only about 20% of the CpGs in CpG islands are methylated. Recent studies on CpG islands suggest that promoters segregate into two classes by CpG content. See, Saxonov et al. PNAS 103(5):1412-7 (2006). [0005] DNA methylation is an epigenetic determinant of gene expression. Patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. [0006] The consequence of methylation is usually gene silencing. DNA methylation also correlates with other cellular processes including embryonic development, chromatin structure, genomic imprinting, somatic X-chromosome inactivation in females, inhibition of transcription and transposition of foreign DNA and timing of DNA replication. When a gene is highly methylated it is less likely to be expressed, possibly because CpG methylation prevents transcription factors from recognizing their cognate binding sites. Proteins that bind methylated DNA may also recruit histone deacetylase to condense adjacent chromatin. Such "closed" chromatin structures prevent binding of transcription factors. Thus the identification of sites in the genome containing 5-meC is important in understanding cell-type specific programs of gene expression and how gene expression profiles are altered during both normal development and diseases such as cancer. Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer caused by increase methylation. [0007] Methylation of cytosine residues in DNA plays an important role in gene regulation. Methylation of cytosine may lead to decreased gene expression by, for example, disruption of local chromatin structure, inhibition of transcription factor-DNA binding, or by recruitment of proteins which interact specifically with methylated sequences and prevent transcription factor binding. DNA methylation is required for normal embryonic development and changes in methylation are often associated with disease. Genomic imprinting, X chromosome inactivation, chromatin modification, and silencing of endogenous retroviruses all depend on establishing and maintaining proper methylation patterns. Abnormal methylation is a hallmark of cancer cells and silencing of tumor suppressor genes is thought to contribute to carcinogenesis. Methylation mapping using microarray-based approaches may be used, for example, to profile cancer cells revealing a pattern of DNA methylation that may be used, for example, to diagnose a malignancy, predict treatment outcome or monitor progression of disease. Methylation in eukaryotes can also function to inhibit the activity of viruses and transposons, see Jones et al. EMBO J. 17:6385-6393 (1998). Alterations in the normal methylation process have also been shown to be associated with genomic instability (Lengauer et al. Proc. Natl. Acad. Sci. USA 94:2545-2550, 1997). Such abnormal epigenetic changes may be found in many types of cancer and can serve as potential markers for oncogenic transformation. SUMMARY OF THE INVENTION [0008] Methods for analyzing the methylation status of cytosines in genomic DNA are disclosed. [0009] In some aspects the methods include a step of multiplex amplification of a plurality of regions of interest. The methods provide for the addition of known priming sequences to the 5' and 3' ends of the sequences to be amplified so that subsequent amplification may be performed using primers to the known priming sequences. Such multiplexed amplification reactions provide high specificity and uniform amplification of templates. [0010] In a first aspect, the invention provides a method for multiplex locus specific amplification of a plurality of templates to provide a plurality of templates with known 5' and 3' ends. [0011] The template may be derived from cDNA or genomic DNA, from a single individual or from a plurality of individuals. The template may, for example, be genomic DNA derived from a eukaryote, such as a human being. [0012] The multiplex methods of the present invention may include at least 10 templates of distinct sequence, at least 100 templates of distinct sequence, at least 1000 templates of distinct sequence, or more. Usefully, at least one of the first and second oligonucleotides comprises a bar code sequence, thus allowing concurrent detection of all amplified templates. [0013] In one embodiment, genomic DNA is modified by bisulfite. Fragments of the modified DNA are generated with defined ends using locus specific primer extension. [0014] The extension products have defined ends and are then hybridized to a dU probe and adaptor sequences are ligated to the ends. The dU probe is degraded and the adaptor ligated fragments are amplified. [0015] In some aspects the methods are used to classify a tissue into a class, for example, a known tumor class. The hybridization pattern obtained from the tissue sample, using the disclosed methods, is compared to hybridization patterns from samples from tissues of known tumor class, obtained using the disclosed methods. [0016] In one aspect a method for analyzing the methylation of a plurality of cytosines in a plurality of target sequences is disclosed. A genomic DNA sample is fragmented to generate fragments that include a mixture of target fragments and non-target fragments. The fragments are mixed with a common primer sequence and a collection of dU probes that are complementary to different target sequences to be analyzed. Each dU probe has a sequence that is complementary to a different target fragment flanked at both ends by the complement of the common primer sequence. Target fragments and common primer sequences hybridize to dU probes to form ligation complexes and ligase is added to ligate the common primer sequences to the target fragments in the ligation complexes. The dU probes are digested using UDG and the ligated products are treated with bisulfite and amplified. The amplification product is analyzed, for example, by hybridization to an array to determine the methylation state of cytosines in the starting sample by detecting sequence changes corresponding to bisulfite modification. [0017] In another aspect DNA is fragmented with a methylation sensitive enzyme so that only unmethylated DNA is fragmented. The overhang created by cleavage is filled in with a DNA polymerase, marking the unmethylated fragments with an additional sequence. The DNA is then fragmented with an isoschizomer of the first enzyme that is methylation insensitive so it will cleave the methylated sites. The fragments are then hybridized to dU probes that are designed to hybridize to either the fragment generated by cleavage then filling or cleavage alone. Amplification products are generated that are differentially detectable, for example, by being marked with different tag sequences. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which like characters refer to like parts throughout, and in which: [0019] FIG. 1 is a schematic representation illustrating methods for appending known sequences to a single-stranded nucleic acid template at specific positions. In FIG. 1A a template with defined ends is obtained by primer extension. In FIG. 1B common sequences are ligated to the ends of the template. FIG. 1C shows an alternate embodiment for digestion of the template probe. [0020] FIG. 2A illustrates one embodiment of a dU probe. Continue reading... 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