Patent ID: 6251594
Filing Date: 2001-06-26
Classification: C07H,C12Q

Abstract:
A method for determining DNA methylation status at a cytosine residue of a CpG sequence, comprising the steps of:(a) obtaining genomic DNA from a DNA sample to be assayed;(b) reacting the genomic DNA with sodium bisulfite to convert unmethylated cytosine residues to uracil residues while leaving any 5-methylcytosine residues unchanged to create an exposed bisulfite-converted DNA sample having binding sites for primers specific for the bisulfite-converted DNA sample;(c) performing a PCR amplification procedure using top strand or bottom strand specific primers;(d) isolating the PCR amplification products;(e) performing a primer extension reaction using a Ms-SNuPE primer, (.sup.32 P) dNTPs and Taq polymerase, wherein the Ms-SNuPE primer comprises from about a 15 mer to about a 22 mer length primer sequence that is complementary to the bisulfite-converted DNA sample and terminates immediately 5' of the cytosine residue of the CpG sequence to be assayed; and(f) determining the methylation status at the cytosine residue of the CpG sequence by measuring the incorporation of different .sup.32 P-labeled dNTPs.