Method for detecting methylation status by using methylation-independent primers

摘要

A reliable and highly sensitive method is provided for detecting methylation status of CpG-containing nucleic acids by nucleic acid amplification and melting curve analysis of amplification products. The methods and compositions employs a novel design of primers, CpG-containing methylation-independent oligonucleotide primers, wherein both unmethylated and methylated alleles of a CpG-containing nucleic acid can be detected by use of only one set of primers after the CpG-containing nucleic acid has been subjected to cytosine to thymine conversion of unmethylated Cytosine. The method is useful for detection of methylation status in for example cancer genes and other disease related genes, wherein methylation influences gene expression.

主权项

1. A method for detecting the methylation status of a CpG-containing nucleic acid in a sample comprising the steps of:
a) providing said sample comprising a CpG-containing nucleic acid, b) denaturing said CpG-containing nucleic acid, thereby producing denatured CpG-containing nucleic acid, c) treating said denatured CpG-containing nucleic acid with bisulphite which modifies at least one unmethylated cytosine in said CpG-containing nucleic acid, thereby producing treated CpG-containing nucleic acid, d) amplifying said treated CpG-containing nucleic acid by means of at least one methylation-independent oligonucleotide primer, which comprises within the first 10 nucleotides of the 5′-end, at least one CpG dinucleotide in a region which hybridizes to the CpG-containing nucleic acid, wherein said at least one methylation-independent oligonucleotide primer hybridizes to both methylated and unmethylated CpG-containing nucleic acids after modification, and e) analyzing the amplified CpG-containing nucleic acid by melting curve analysis.