Fast genotyping of K-RAS codons 12 and 13 based on streptavidin magnetic microbeads equipped with biotin-coupled single base-pair mismatch PCR (SM-PCR)

In this study, a single base-pair mismatch polymerase chain reaction (SM-PCR) combined with streptavidin magnetic microbeads (MB) for genotyping of codons 12 and 13 of K-RAS gene was established. It was performed by adding three primers including codon 12 forward single-base mismatch primer, codon 13 forward single-base mismatch primer and one universal reverse primer to differentiate single nucleotide polymorphisms. The both forward primers were designed to carry a single-base mismatch nucleotide to either codons 12 and 13. This design was included to increase the primer specificity and to enhance the discrimination power of the point mutation. Two restriction enzymes (StyD4I and HaeIII) were available for double digestion of codons 12 and 13 at the single tube. The wild type of codon 12 and 13 was digested, but the mutant type was not. And then, using the magnetic microbeads for purifying. Finally, wild type of codon 12 and 13 have different emission wavelength, but mutant type did not due to no digestion. This method showed a good consistency between genotyping results and direct sequencing results. The SM-PCR combined with MB method was feasible and efficient for screening of K-RAS codons 12 and 13 in populations.