High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type alleles.

The missense mutations at codons 12 and 13 of KRAS gene have been confirmed as a predictor of nonresponse to EGFR-targeted therapy with monoclonal antibodies cetuximab and panitumumab in patients with metastatic colorectal carcinoma (mCRC). Because of the intra-tumor heterogeneity at genetic levels, it is important to develop sensitive and selective assays to detect above KRAS mutation of rare mutated cells in the presence of large excess of wild-type cells. In the present study, wild-type blocking PCR (WTB-PCR) was developed to detect the aforementioned KRAS mutations, in which a chimera composed of locked nucleic acid (LNA) and DNA was used to inhibit with high sensitivity the amplification of wild-type KRAS alleles whereas it allowed the highly selective amplification of mutated KRAS alleles. Using mutated KRAS from HCT-116 as spiking DNA, the results showed that WTB-PCR could detect mutated alleles in a ratio of 1:10,000 (i.e., 0.01%) wild-type alleles and at a single copy level. For its further applications to detect aforementioned KRAS mutations in 20 cases of mCRC patients, the results showed that the detected mutation percentage of WTB-PCR (60%, 12/20) was higher than that of traditional PCR (45%, 9/20). Moreover, two patients respectively having synonymous mutated codons 13 (i.e., c.39C > T) and missense mutated codons 14 (i.e., c.40G > A) could be also only detected by WTB-PCR. In conclusion, the current WTB-PCR was a rapid, simple, and low-cost method to detect a trace amount of mutated KRAS gene.