A 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. Patent 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. In a standard procedure, a cocktail of standards for a plurality of genes of interest (TARGETgene) is added to a sample of serum. Once total DNA is purified and processed, a PCR (multiplex step) is performed wherein the STDgene and the TARGETgene are co-amplified with the same external primer set. In the second nested PCR step, amplicons present in a dilution of the first PCR reaction are subjected to real time PCR, and quantified for each gene in one well by two-color real-time PCR. Products are calculated by absolute quantitation with internal primer sets specific for the methylated TARGETgene and associated STDgene. Methods of making the STDgene standards and the use of the cMethDNA methods and kits containing the same are disclosed.