Detection of low-level DNA mutation by ARMS-blocker-Tm PCR

Low-level DNA mutations play important roles in cancer prognosis and treatment. However, most existing methods for the detection of low-level DNA mutations are insufficient for clinical applications because of the high background of wild-type DNA. In this study, a novel assay based on Tm-dependent inhibition of wild type template amplification was developed. The defining characteristic of this assay is an additional annealing step was introduced into the ARMS-blocker PCR. The temperature of this additional annealing step is equal to the Tm of the blocker. Due to this additional annealing step, the blocker can preferentially and specifically bind the wild-type DNA. Thus, the inhibition of wild type template is realized and the mutant DNA is enriched. The sensitivity of this assay was between 10−4 and 10−5, which is approximately 5 to 10 times greater than the sensitivity of the assay without the additional annealing step. To evaluate the performance of this assay in detecting K-ras mutation, we analyzed 100 formalin-fixed paraffin-embedded (FFPE) specimens from colorectal cancer patients using this new assay and Sanger sequencing. Of the clinical samples, 27 samples were positive for K-ras mutation by both methods. Our results indicated that this new assay is a highly selective, convenient, and economical method for detecting rare mutations in the presence of higher concentrations of wild-type DNA. •In this study, a novel assay based on Tm-dependent inhibition of wild type template amplification was developed.•The defining characteristic of this assay is that an additional annealing step was introduced into the ARMS-blocker PCR.•The temperature of this additional annealing step is equal to the Tm of the blocker.•Due to this additional annealing step, the blocker can preferentially and specifically bind the wild-type DNA.•Thus, the inhibition of wild type template is realized and the mutant DNA is enriched.