Quantitative multiplex methylation specific PCR method—cMethDNA, reagents, and its use

摘要

The cMethDNA method of the present invention is a novel modification of the QM-MSP method (U.S. Pat. No. 8,062,849), specifically intended to quantitatively detect tumor DNA (or other circulating DNAs) in fluids such as serum or plasma at the lowest copy number yet reported. Unique compared to any other PCR-based assay, a small number of copies of a synthetic polynucleotide standard (STDgene) is added to an aliquot of patient serum with standards for a plurality of genes of interest (TARGETgene). Once total DNA is purified PCR is performed wherein the STDgene and the TARGETgene are co-amplified with the same external primer set, and the amplicons present in a dilution of the first PCR reaction are subjected to real time PCR, and quantified for each gene. Methods of making the STDgene standards and the use of the cMethDNA methods and kits containing the same are disclosed.