<PAPERS and REPORTS> Quantitative PCR Discriminating Point Mutation of KRAS Gene Using Chemically Modified Primers

Complete allele specific PCR was achieved by 2’-OMeRNA modified primer and HiDi DNA PolymeraseTM (myPOLS Biotec GmbH). PCR reactions with primers modified with 2’-OMeRNA at the third position from the 3’-end and a mutation point at the 3’-end showed complete discrimination of KRASwt(35G) and KRASG12D(35A). Namely, PCR with the primer POM3g which has 2’-OMeRNA at the third position from the 3’-end and normal G at the 3’-end amplified the KRASwt(35G) template with a Ct value of 25.05 &#177; 0.14, but did not amplify the mutant KRASG12D(35A) template at all. On the other hand, PCR with the primer POM3a which has 2’-OMeRNA at the third position from the 3’-end and normal A at the 3’-end amplified the KRASG12D(35A) template with a Ct value of 24.79 &#177; 0.07, but did not amplify the mutant KRASwt(35G) template at all. To the best of our knowledge, this is the first example of a complete allele specific PCR and promising for the application to the diagnostic panel for cancer.