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  Quantitative methylation detection in dna samplesUSPTO Application #: 20060088866Title: Quantitative methylation detection in dna samples Abstract: Described is a method for methylation detection in a DNA sample. An isolated genomic DNA sample is treated in a manner capable of distinguishing methylated from unmethylated cytosine bases. The pretreated DNA is amplified using at least one oligonucleotide primer, a polymerase and a set of nucleotides of which at least one is labeled with a first type of label. A sequence-specific oligonucleotide probe, marked with a second type of label, hybridizes to the amplification product and a FRET reaction occurs if a labeled oligonucleotide is present in close proximity in the amplification product. The method determines the level of methylation of a sample by measuring the extent of fluorescence resonance energy transfer (FRET) between the donor and acceptor fluorophore. (end of abstract) Agent: Kriegsman & Kriegsman - Framingham, MA, US Inventor: Susan Cottrell USPTO Applicaton #: 20060088866 - 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 20060088866. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation of U.S. patent application Ser. No. 10/068,553, filed Feb. 6, 2002, which is incorporated herein by reference. FIELD OF THE INVENTION [0002] This invention relates to the analysis of nucleic acids, especially to the analysis of methylation patterns in genomic DNA by providing a means of detecting nucleotides, that are characteristic for methylated sites after bisulfite treatment of the genomic DNA. The method utilises the incorporation of labels and the detection of fluorescence resonance energy transfer (FRET) of the amplified sample DNA. PRIOR ART DNA Methylation [0003] The levels of observation that have been studied in recent years in molecular biology have concentrated on genes, the translation of those genes into RNA, and the transcription of the RNA into protein. There has been a more limited analysis of the regulatory mechanisms associated with gene control. Gene regulation, for example, at what stage of development of the individual a gene is activated or inhibited, and the tissue specific nature of this regulation is less understood. However, it can be correlated with a high degree of probability to the extent and nature of methylation of the gene or genome. From this observation it is reasonable to infer that pathogenic genetic disorders may be detected from irregular genetic methylation patterns. [0004] The efforts of the Human Genome project are concentrated on the sequencing of the human genome. It is expected that this will yield considerable therapeutic and diagnostic benefits for the treatment of disease. However, these efforts have so far been unable to address a significant aspect of genetic disorders, the epigenetic factor. The epigenetic regulation of gene transcription has been shown to effect many disorders. One of the most significant epigenetic mechanisms so far identified has been the methylation of cytosine. The methylation of cytosine at the 5 position is the only known modification of genomic DNA. Although the exact mechanisms by which DNA methylation effects DNA transcription are unknown, the relationship between disease and methylation has been well documented. In particular methylation patterns of CpG islands within regulatory regions of genome appear to be highly tissue specific. Therefore, it follows that misregulation of genes may be predicted by comparing their methylation pattern with phenotypically `normal` expression patterns. The following are cases of disease associated with modified methylation patterns. [0005] Head and neck cancer (Sanchez-Cespedes M et al. "Gene promoter hypermethylation in tumors and serum of head and neck cancer patients" Cancer Res. 2000 Feb. 15; 60 (4):892-5) [0006] Hodgkin's disease (Garcia J F et al "Loss of p16 protein expression associated wiht methylation of the p16INK4A gene is a frequant finding in Hodgkin's disease" Lab invest 1999 December; 79 (12):1453-9) [0007] Gastric cancer (Yanagisawa Y et al., "Methylation of the hMLH1 promoter in familial gastric cancer with microsatellite instability" Int J Cancer 2000 Jan. 1; 85 (1):50-3) [0008] Prader-Willi/Angelman's syndrome (Zeschnigh et al "Imprinted segments in the human genome: different DNA methylation patterns in the Prader Willi/Angelman syndrome region as determined by the genomic sequencing method" Human Mol. Genetics (1997) (6) 3 pp 387-395) [0009] ICF syndrome (Tuck-Muller et al "CMDNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients" Cytogenet Call Genet 2000; 89(1-2):121-8 [0010] Dermatofibroma (Chen T C et al "Dermatofibroma is a clonal proliferative disease" J Cutan Pathol 2000 January; 27 (1):36-9) [0011] Hypertension (Lee S D et al., "Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension" J clin Invest 1998 Mar. 1, 101 (5):927-34) [0012] Autism (Klauck S M et al., "Molecular genetic analysis of the FMR-1 gene in a large collection of autistic patients" Human Genet 1997 August; 100 (2): 224-9) [0013] Fragile X Syndrome (Hornstra I K et al., "High resolution methylation analysis of the FMR1 gene trinucleotide repeat region in fragile X syndrome" Hum Mol Genet 1993 October, 2(10):1659-65) [0014] Huntigton's disease (Ferluga J et al. "possible organ and age related epigenetic factors in Huntington's disease and colorectal carcinoma" Med hyptheses 1989 May; 29(1);51-4 [0015] All of the above documents are hereby incorporated by reference. Bisulphite Treatment [0016] A relatively new and currently the most frequently used method for analyzing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behaviour. However, 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, can now be detected as the only remaining cytosine using "normal" molecular biological techniques, for example, by amplification and hybridisation or sequencing. All of these techniques are based on base pairing which can now be fully exploited. In terms of sensitivity, the prior art is defined by a method which encloses the DNA to be analysed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Using this method, it is possible to analyse individual cells, which illustrates the potential of the method. However, currently only individual regions of a length of up to approximately 3000 base pairs are analysed, a global analysis of cells for thousands of possible methylation events is not possible. However, this method cannot reliably analyse very small fragments from small sample quantities either. These are lost through the matrix in spite of the diffusion protection. [0017] An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255. [0018] To date, barring few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 March-April; 5(2):94-8) the bisulfite technique is only used in research. Always, however, short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment and either completely sequenced (Olek A, Walter J. The preimplantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 November; 17(3):275-6) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L, Jones P A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO Patent 9500669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15; 25(12):2532-4). In addition, detection by hybridisation has also been described (Olek et al., WO 99 28498). [0019] Further publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 June; 16(6):431-6, 431; zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb. 25; 22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 51 region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97 46705, WO 95 15373 and WO 45560. Continue reading... Full patent description for Quantitative methylation detection in dna samples Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Quantitative methylation detection in dna samples patent application. 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