ANALYSIS OF METHYLATION PATTERNS IN GENOMIC DNA USING BISULFITE TREATMENT AND FLUORESCENCE POLARISATION ASSAY TECHNIQUES

摘要

A method for the analysis of the methylation of cytosine bases in genomic DNA samples, comprising the following steps:(a) the genomic DNA is chemically treated in such a manner that cytosine is converted into uracil or a similar base regarding the base pairing behaviour in the DNA duplex, 5 methylcytosine however remains unchanged;(b) the chemically treated DNA is amplified using of at least one species of oligonucleotide (type A) as a primer in a polymerase reaction;(c) the amplificate is left in solution with one or more species of fluorophore labelled nucleotides and one or more species of oligonucleotide (type B) , wherein the type B oligonucleotide hybridises under appropriate conditions with its 3' end directly on or up to 10 bases from the position to be examined, and wherein said type B oligonucleotide is at least partly nuclease resistant;(d) the hybridised oligonucleotide (type B) is extended by means of a polymerase by at least one nucleotide, whereby the extension is dependant upon the methylation status of the respective cytosine position in the genomic DNA sample;(e) the solution is incubated with a phosphodiesterase, which is capable of digesting nucleic acids, however incompletely digests the type B oligonucleotides and its extension products;(f) the fluorescence polarisation of the solution is measured whereby for each fluorescent label used one determines the degree of polarisation.