REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS
https://doi.org/10.21294/1814-4861-2018-17-4-30-35
Abstract
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.
About the Authors
V. A. Shamanin
Federal Research Center of Fundamental and Translational Medicine
Biolink Ltd
Russian Federation
Russian Federation
2, Timakova Street, 630117-Novosibirsk, Russia
2, Timakova Street, 630117-Novosibirsk, Russia
PhD, Senior Researcher, Federal Research Center of Fundamental and Translational Medicine
I. V. Karpov
Federal Research Center of Fundamental and Translational Medicine
Biolink Ltd
Russian Federation
Russian Federation
2, Timakova Street, 630117-Novosibirsk, Russia
2, Timakova Street, 630117-Novosibirsk, Russia
laboratory assistant, Federal Research Center of Fundamental and Translational Medicine
E. E. Pisareva
Federal Research Center of Fundamental and Translational Medicine
Russian Federation
Russian Federation
2, Timakova Street, 630117-Novosibirsk, Russia
PhD, Senior Researcher, Federal Research Center of Fundamental and Translational Medicine
N. I. Gutkina
Federal Research Center of Fundamental and Translational Medicine
Russian Federation
Russian Federation
2, Timakova Street, 630117-Novosibirsk, Russia
PhD, Senior Researcher, Federal Research Center of Fundamental and Translational Medicine
S. P. Kovalenko
Federal Research Center of Fundamental and Translational Medicine
Biolink Ltd
Russian Federation
Russian Federation
2, Timakova Street, 630117-Novosibirsk, Russia
2, Timakova Street, 630117-Novosibirsk, Russia
DSc, Head of Laboratory, Federal Research Center of Fundamental and Translational Medicine
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Review
For citations:
Shamanin V.A., Karpov I.V., Pisareva E.E., Gutkina N.I., Kovalenko S.P. REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS. Siberian journal of oncology. 2018;17(4):30-35. https://doi.org/10.21294/1814-4861-2018-17-4-30-35
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