REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS
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 m...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | Russian |
| Published: |
Russian Academy of Sciences, Tomsk National Research Medical Center
2018-09-01
|
| Series: | Сибирский онкологический журнал |
| Subjects: | |
| Online Access: | https://www.siboncoj.ru/jour/article/view/812 |
| _version_ | 1797875853123649536 |
|---|---|
| author | V. A. Shamanin I. V. Karpov E. E. Pisareva N. I. Gutkina S. P. Kovalenko |
| author_facet | V. A. Shamanin I. V. Karpov E. E. Pisareva N. I. Gutkina S. P. Kovalenko |
| author_sort | V. A. Shamanin |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T01:53:11Z |
| format | Article |
| id | doaj.art-4c0606961de94c8084c06cd29a8b52a0 |
| institution | Directory Open Access Journal |
| issn | 1814-4861 2312-3168 |
| language | Russian |
| last_indexed | 2024-04-10T01:53:11Z |
| publishDate | 2018-09-01 |
| publisher | Russian Academy of Sciences, Tomsk National Research Medical Center |
| record_format | Article |
| series | Сибирский онкологический журнал |
| spelling | doaj.art-4c0606961de94c8084c06cd29a8b52a02023-03-13T09:05:51ZrusRussian Academy of Sciences, Tomsk National Research Medical CenterСибирский онкологический журнал1814-48612312-31682018-09-01174303510.21294/1814-4861-2018-17-4-30-35558REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONSV. A. Shamanin0I. V. Karpov1E. E. Pisareva2N. I. Gutkina3S. P. Kovalenko4Федеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр фундаментальной и трансляционной медицины» ООО «БиоЛинк»Федеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр фундаментальной и трансляционной медицины» ООО «БиоЛинк»Федеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр фундаментальной и трансляционной медицины»Федеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр фундаментальной и трансляционной медицины»Федеральное государственное бюджетное научное учреждение «Федеральный исследовательский центр фундаментальной и трансляционной медицины» ООО «БиоЛинк»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.https://www.siboncoj.ru/jour/article/view/812запертая нуклеиновая кислотазажим pcrмутацииkrasbrafднк-полимераза taq |
| spellingShingle | V. A. Shamanin I. V. Karpov E. E. Pisareva N. I. Gutkina S. P. Kovalenko REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS Сибирский онкологический журнал запертая нуклеиновая кислота зажим pcr мутации kras braf днк-полимераза taq |
| title | REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS |
| title_full | REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS |
| title_fullStr | REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS |
| title_full_unstemmed | REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS |
| title_short | REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS |
| title_sort | requirements for efficient pcr clamping by locked nucleic acid oligonucleoties for simple and sensitive detection of somatic mutations |
| topic | запертая нуклеиновая кислота зажим pcr мутации kras braf днк-полимераза taq |
| url | https://www.siboncoj.ru/jour/article/view/812 |
| work_keys_str_mv | AT vashamanin requirementsforefficientpcrclampingbylockednucleicacidoligonucleotiesforsimpleandsensitivedetectionofsomaticmutations AT ivkarpov requirementsforefficientpcrclampingbylockednucleicacidoligonucleotiesforsimpleandsensitivedetectionofsomaticmutations AT eepisareva requirementsforefficientpcrclampingbylockednucleicacidoligonucleotiesforsimpleandsensitivedetectionofsomaticmutations AT nigutkina requirementsforefficientpcrclampingbylockednucleicacidoligonucleotiesforsimpleandsensitivedetectionofsomaticmutations AT spkovalenko requirementsforefficientpcrclampingbylockednucleicacidoligonucleotiesforsimpleandsensitivedetectionofsomaticmutations |