PCR clamping/wild-type blocking PCR with non-extendable locked nucleic acid (LNA) oligonucleotides is used for sensitive detection of somatic mutations in tumors. Various versions of the technique use different DNA polymerases and LNA oligonucleotides with and without additional phosphorothioate modifications. Here we studied requirements for successful PCR clamping with LNA oligonucleotides and Taq DNA polymerase for analysis of mutations in KRAS and BRAF genes by means of real-time PCR and Sanger sequencing. We found that addition of phosphorothioate linkages at the 5’-end of LNA oligonucleotide to protect from 5’- exonuclease activity of Taq DNA polymerase did not improve clamping. For most target sequences, efficient clamping was observed at melting temperature of LNA oligonucleotide 20‑25°C above annealing/extension temperature of the PCR with a 2-step protocol. Under such conditions, simple and sensitive detection of mutations in KRAS and BRAF genes was feasible using real-time PCR with TaqMan probes or Sanger sequencing.
Highly Sensitive Detection of Dengue Virus Nucleic Acid in Samples from Clinically Ill Patients
