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2013 | 60 | 1 | 57-64
Article title

ARMS-PCR for detection of BRAF V600E hotspot mutation in comparison with Real-Time PCR-based techniques

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Abstracts
EN
BRAF mutation testing is one of the best examples how modern genetic testing may help to effectively use targeted therapies in cancer patients. Since many different genetic techniques are employed to assess BRAF mutation status with no available comparison of their sensitivity and usefulness for different types of samples, we decided to evaluate our own PCR-based assay employing the amplification refractory mutation system (ARMS-PCR) to detect the most common hotspot mutation c. T1799A (p. V600E) by comparing it with two qPCR based assays: a commercially available test with hybridizing probes (TIB MOLBIOL) and high resolution melting (HRM). Positive results were verified with Sanger sequencing. DNA from two cancer cell lines with known mutation status and from tissue samples from melanoma and gastric cancer was used. ARMS-PCR was the most sensitive method with the level of detection of the mutant allele at 2%. Similar sensitivity was observed for the qPCR-based commercial test employing hybridizing probes; however, this test cannot exclude negative results from poor or low quality samples. Another qPCR-based method, HRM, had lower sensitivity with the detection level of approximately 20%. An additional drawback of HRM methodology was the inability to distinguish between wild type and mutant homozygotes in a straightforward assay, probably due to the character of this particular mutation (T\>A). Sanger sequencing had the sensitivity of the detection of mutant allele similar to HRM, approx. 20%. In conclusion, simple ARMS-PCR may be considered the method of choice for rapid, cost-effective screening for BRAF p. V600E mutation.
Publisher

Year
Volume
60
Issue
1
Pages
57-64
Physical description
Dates
published
2013
received
2012-08-16
revised
2012-12-15
accepted
2013-01-14
(unknown)
2013-03-05
Contributors
  • Department of Immunology, Medical University of Warsaw, Warsaw, Poland
  • Department of Immunology, Medical University of Warsaw, Warsaw, Poland
  • Department of Chemotherapy, The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland
author
  • Department of Medical Genetics, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland
  • Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland
  • Department of Immunology, Medical University of Warsaw, Warsaw, Poland
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Document Type
Publication order reference
Identifiers
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv60p57kz
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