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A Comparison Between Full-COLD PCR/HRM and PCR Sequencing for Detection of Mutations in Exon 9 of PIK3CA in Breast Cancer Patients

Author:
Ghalamkari, Safoura, Khosravian, Farinaz, Mianesaz, Hamidreza, Kazemi, Mohammad, Behjati, Mohaddeseh, Hakimian, Sayyed Mohammadreza, Salehi, Mansoor
Source:
Applied biochemistry and biotechnology 2019 v.187 no.3 pp. 975-983
ISSN:
0273-2289
Subject:
DNA, breast neoplasms, carcinogenesis, cell lines, cost effectiveness, denaturation, exons, melting, mutants, patients, polymerase chain reaction, screening, sequence analysis, somatic mutation, temperature
Abstract:
One of the most common somatic mutations in breast cancer is found in PIK3CA with a prevalence rate of 18–45%. Different variants of this gene are considered as resistance markers for treatment with HER2-targeted medicines. Conventional molecular methods such as Sanger sequencing are not able to detect mutations with low abundance in a mixture of wild-type DNA, especially in the early stages of cancer development. In this study, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM) were combined for detection of mutations in exon 9 of PIK3CA; DNA, therefore, was extracted from MCF-7 and BT-474 as mutant and wild-type cell lines respectively. Thereafter, serial dilutions of extracted DNA were used to determine sensitivity of full-COLD PCR/HRM in comparison with conventional PCR sequencing as the gold standard method. Cell line experiments resulted in almost 30 fold increase in sensitivity by use of full-COLD PCR/HRM. In addition, 40 patients with primary breast cancer were investigated with the mentioned methods. As a result of this part of study, four mutations were detected by conventional PCR sequencing including E542K and E545K mutations in three and one samples respectively. Whereas, full-COLD PCR/HRM was able to detect one E542K mutation more than gold standard method which caused the percentage of sensitivity to get improved by 2.5% (10 to 12.5%). Our results clearly demonstrated that full-COLD PCR/HRM could detect lower levels of mutations in wild-type background as a sensitive method with simple and cost-effective procedure; therefore, it can prospectively be used in screening of patients with early-stage breast cancers.
Agid:
6354507