Sensitive and selective detections of codon 12 and 13 KRAS mutations in a single tube using modified wild-type blocker.

It was hypothesized that in the WTB-PCR system, the greater number of cycles, associated with the thermodynamic driving force of DNA polymerase resulted in artificial introduction of mutant nucleotides in amplicons. In the current study, universal WTB-PCR was developed to overcome these limitations, in which two strategies were used: phosphorothioate modifications were made at the 5'-termini bases of the WTB oligonucleotides, and amplification of referenced internal positive controller (RIPC) fragments was performed. The results showed that universal WTB-PCR could detect single-copy KRAS mutant alleles with higher selectivity (i.e., 0.01%), and with greater ability to eliminate non-specific amplification of KRAS wild-type alleles in amounts up to 200 ng. Moreover, the introduction of referenced internal positive controller (RIPC) fragments prevented false-negative results caused by inadequate amounts of input sample DNA, and allowed for quantitative analysis of the mutation levels in each FFPE sample. In clinical application in 50 samples of FFPE tissue sections from mCRC patients, 70% (35/50) showed various mutations at codons 12 and 13 of KRAS genes; 30% (15/50) could be detected by traditional PCR without WTB oligonucleotides. In conclusion, universal WTB-PCR is a rapid, simple and low-cost method for detection of low-abundance KRAS mutations in mCRC patients.