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Technique / Molecular Biology / DNA analysis techniques / DNA methylation
| DNA methylation methods include: The methylation-sensitive single-nucleotide primer extension (Ms-SNuPE), combined bisulfite restriction analysis (COBRA), bisulfite treatment and PCR-single-strand conformation polymorphism analysis (Bisulfite-PCR-SSCP/BiPS), Methylation-specific PCR, Bisulfite sequencing. |
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Methylation and uracil interference assays for analysis of protein-DNA interactions. (Curr Protoc Mol Biol. 2001 May;Chapter 12:Unit 12.3.) Baldwin AS Jr, Oettinger M, Struhl K. University of North Carolina, Chapel Hill, North Carolina, USA. Interference assays identify specific residues in the DNA binding site that, when modified, interfere with binding of the protein. The protocols use end-labeled DNA probes that are modified at an average of one site per molecule of probe. These probes are incubated with the protein of interest, and protein-DNA complexes are separated from free probe by the mobility shift assay. A DNA probe that is modified at a position that interferes with binding will not be retarded in this assay; thus, the specific protein-DNA complex is depleted for DNA that contains modifications on bases important for binding. After gel purification, the bound and unbound DNA are specifically cleaved at the modified residues and the resulting products analyzed by electrophoresis on polyacrylamide sequencing gels and autoradiography. In the methylation interference protocol presented here, probes are generated by methylating guanines (at the N-7 position) and adenines (at the N-3 position) with DMS; these methylated bases are cleaved specifically by piperidine. In the uracil interference protocol, probes are generated by PCR amplification in the presence of a mixture of TTP and dUTP, thereby producing products in which thymine residues are replaced by deoxyuracil residues (which contains hydrogen in place of the thymine 5-methyl group). Uracil bases are specifically cleaved by uracil-N-glycosylase to generate apyrimidinic sites that are susceptible to piperidine. These procedures provide complementary information about the nucleotides involved in protein-DNA interactions. 
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Last update: 
16-May-2008 06:36 pm
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HOXA11 DNA methylation-A novel prognostic biomarker in ovarian cancer. Int J Cancer. 2008 May 13;
Authors: Fiegl H, Windbichler G, Mueller-Holzner E, Goebel G, Lechner M, Jacobs IJ, Widschwendter M
Epigenetic alterations play a major role in cancer. Recently we reported that stem cell Polycomb group targets (PcGTs) are up to 12-fold more likely to have cancer-specific promoter DNA hypermethylation than nontargets. To identify potential, prognostic DNA methylation markers in ovarian cancer we analyzed the DNA methylation at 71 different loci in 22 ovarian cancers and 18 non-neoplastic ovarian specimens by means of a quantitative, real-time PCR-based technique (MethyLight). We identified DNA methylation of HOXA10 and HOXA11, both of them PcGTs, to be the best discriminators between cancer and non-neoplastic tissue. In an independent set consisting of 92 ovarian cancer specimens further analysis demonstrated that HOXA11 DNA methylation is (i) strongly associated with the residual tumor after cytoreductive surgery and (ii) is a marker indicating poor prognosis. HOXA11 DNA methylation was independently associated with poor outcome [relative risk for death 3.4 (95% CI 1.2-9.9; p = 0.03)]. These findings support the view that the technical inability to optimally cytoreduce ovarian cancer is associated with particular molecular alterations in the tumor which per se define a subgroup of patients with poor outcome. (c) 2008 Wiley-Liss, Inc.
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