A novel mutant allele specific amplification and electrochemiluminescence method for the detection of point mutation in clinical samples

A novel mutant allele specific amplification (MASA) and electrochemiluminescence (ECL) method for point mutation detection is proposed. Briefly, the target gene was amplified by a biotinylated mutant specific sense primer and a Ru(bpy)32+ (TBR)-labeled universal antisense primer. Only the mutant allele can be selectively amplified by the mutant specific primer pair. Then, the MASA product was captured onto the streptavidinylated magnetic beads through biotin–streptavidin linkage and detected by measuring the ECL emission of TBR. The method was applied to detect a possible point mutation at codon 12 of K-ras oncogene in 30 colorectal cancer (CAC) clinical samples. The experimental results show that the method can detect K-ras mutant in a 5000-fold excess of wild-type allele. Furthermore, different kinds of mutations can be clearly discriminated. The point mutation was found in 15 (50%) out of 30 CAC samples. This novel MASA–ECL method could potentially become a sensitive, specific, simple, rapid and safe approach for point mutation detection.