An ECL-PCR method for quantitative detection of point mutation

摘要

A new method for identification of point mutations was proposed. Polymerase chain reaction (PCR) amplificationof a sequence from genomic DNA was followed by digestion with a kind of restriction enzyme, which only cut thewild-type amplicon containing its recognition site. Reaction products were detected by electrochemiluminescence (ECL)assay after adsorption of the resulting DNA duplexes to the solid phase. One strand of PCR products carries biotin to bebound on a streptavidin-coated microbead for sample selection. Another strand carries Ru(bpy)_3~(2+) (TBR) to react withtripropylamine (TPA) to emit light for ECL detection. The method was applied to detect a specific point mutation inH-ras oncogene in T24 cell line. The results show that the detection limit for H-ras amplicon is 100 fmol and the linearrange is more than 3 orders of magnitude, thus, make quantitative analysis possible. The genotype can be clearlydiscriminated. Results of the study suggest that ECL-PCR is a feasible quantitative method for safe, sensitive and rapiddetection of point mutation in human genes.