Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes
- Авторы: Botezatu I.V.1, Panchuk I.O.1, Stroganova A.M.1, Senderovich A.I.1, Kondratova V.N.1, Shelepov V.P.1, Lichtenstein A.V.1
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Учреждения:
- Research Institute of Carcinogenesis
- Выпуск: Том 51, № 1 (2017)
- Страницы: 41-48
- Раздел: Genomics. Transcriptomics
- URL: https://journals.rcsi.science/0026-8933/article/view/162958
- DOI: https://doi.org/10.1134/S002689331701006X
- ID: 162958
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Аннотация
Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin- fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.
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Об авторах
I. Botezatu
Research Institute of Carcinogenesis
Email: alicht@mail.ru
Россия, Moscow, 115478
I. Panchuk
Research Institute of Carcinogenesis
Email: alicht@mail.ru
Россия, Moscow, 115478
A. Stroganova
Research Institute of Carcinogenesis
Email: alicht@mail.ru
Россия, Moscow, 115478
A. Senderovich
Research Institute of Carcinogenesis
Email: alicht@mail.ru
Россия, Moscow, 115478
V. Kondratova
Research Institute of Carcinogenesis
Email: alicht@mail.ru
Россия, Moscow, 115478
V. Shelepov
Research Institute of Carcinogenesis
Email: alicht@mail.ru
Россия, Moscow, 115478
A. Lichtenstein
Research Institute of Carcinogenesis
Автор, ответственный за переписку.
Email: alicht@mail.ru
Россия, Moscow, 115478
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