Method for monitoring the bisulfite-mediated conversion of DNA

摘要

The present invention relates to a method for monitoring the progression of the bisulfite-mediated conversion of DNA during DNA methylation analysis. The method is based on the reaction of the enzyme uracil-DNA-glycosylase (UNG) with at least one labeled DNA reporter molecule, the reporter molecule comprising at least one unmethylated cytosine residue in its sequence. After bisulfite-mediated conversion of unmethylated cytosine residues in uridin residues UNG removes the uracil bases from the DNA backbone, thus making it susceptible to heat-induced hydrolytic cleavage. Finally, the labels released from the DNA reporter molecule during this fragmentation process are detected.

主权项

1. Method for monitoring the bisulfite-mediated conversion of DNA during DNA methylation analysis, comprising:
(a) providing a sample DNA to be analyzed and at least one DNA reporter molecule, wherein the at least one DNA reporter molecule comprises: (i) in its nucleotide sequence at least one unmethylated cytosine residue; and (ii) at least one label; and wherein the sample DNA and the at least one DNA reporter molecule are provided in spatially separated reaction compartments that are in fluid connection with each other; (b) adding a bisulfite salt to the spatially separated reaction compartments, thus mediating the conversion of the unmethylated cytosine residues comprised in the nucleotide sequences of the sample DNA and the at least one DNA reporter molecule into uridine residues; (c) adding an uracil-DNA-glycosylase to the at least one DNA reporter molecule, thus mediating the removal of the uracil bases obtained in step (b) from the DNA backbone; (d) fragmentation of the DNA obtained in step (c) by heat treatment; and (e) detecting the at least one label released from the at least one DNA reporter molecule during step (d).