Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS.

Mutant KRAS proto-oncogene GTPase (KRAS) serves an important role in predicting the development, diagnosis, treatment and efficacy of targeted drug therapies for colorectal cancer. To improve the detection efficacy of trace amount of mutant KRAS, the locked nucleic acid-based method was modified in the present study. Internal competitive amplification fragments were used to improve the inhibition of wild-type KRAS with a wild-type blocking (WTB) probe and specifically amplify the trace amounts of mutant KRAS. The modified method, quantitative clamp-based polymerase chain reaction technology using WTB coupled with internal competitive reference to enhance the amplification specificity, named WIRE-PCR, completely blocked the amplification of wild-type KRAS in 50-150 ng DNA templates. The added internal competitive amplified fragments were amplified together with the target gene, which were used to reduce base mismatch due to the high number of cycles in PCR and quantify the total amount of DNA. The results demonstrated that WIRE-PCR facilitated the detection of mutated alleles at a single molecular level. In the colorectal biopsies from 50 patients with suspected colorectal cancer, 18 cases (36%) contained mutant KRAS, and the amount of mutant DNA accounted for 18.6-64.2% of the total DNA. WIRE-PCR is a simple, rapid and low-cost quantitative analysis method for the detection of trace amounts of the mutant KRAS.