Evaluation and comparison of C.524G>A mutation detection in TP53 gene by two methods of COLD- PCR/HRM and PCR-Sequencing in different dilutions of DNA mutations

Background and Objectives: According to studies and cosmic database, mutation in TP53 gene is one of the most common somatic mutations in breast cancer which accounts for about 26% of mutations. The C.524G> A mutation is one of the most important somatic mutations in the TP53 gene in breast cancer. One of the reasons for the difficult diagnosis of cancer in early stages is the heterogeneity of tumor tissue. For this purpose, selective amplification and enrichment of small amount of mutated DNA is carried out in the wild-type DNA in COLD-PCR. In this method, by determining and using the critical temperature Tc, mutated DNA fragments are exclusively sequenced and amplified, thereby increasing the amount of mutated DNA in the background of non-mutated DNA. In this study, high resolution melting curve (HRM) method is used to detect and identify the desired mutation.Materials and Methods: SKBR-9 and BT-474 cells were first cultured as mutant and non-mutant cell lines and after DNA extraction, serial dilutions of mutant DNA were prepared by wild-type DNA. All factors of COLD-PCR were adjusted by serial dilutions and sensitivity of COLD-PCR / HRM, PCR / Sequencing, PCR / HRM and COLD-PCR / Sequencing methods for C.524G> A mutation in TP53 gene Evaluated and evaluated.Results: Detection of mutation rate by COLD-PCR / HRM method was possible up to 0.4% dilution of mutated DNA, while PCR / HRM assay was about 6.2% and sensitivity of PCR sequencing method. In this study, the rate of sensitivity of this method was up to 1.5% of the mutated DNA by COLD-PCR prior to PCR Sequencing.

کلیدواژه ها:

Breast cancer ، TP53 gene ، C.524G> A (P.R175H) mutation ، COLD-PCR / HRM ، PCR Sequencing