Comparison of Two Methods of Ice-COLD HRM-PCR and Full-COLD HRM-PCR in Detection of E542K Mutation in PIK3CA gene in Breast Cancer

Introduction & Objectives: The most common cancer through women is breast cancer. Genetic mutations play a major role in the development of breast cancer. About 5% to 10% of cases of breast cancer are inherited, and most cases are reported by somatic mutations. According to the COSMIC database, one of the most important mutations in PIK3CA gene is E542K located in exon 9.Materials & Methods: Co-amplification at lower denaturation temperature PCR (Cold PCR) is a technique that selectively enriches the mutated DNA in the background of healthy DNA. In this method, by using the critical denaturation temperature )TC(temperature in the polymerase chain reaction and choosing the WT-mutant type heteroduplex denaturation, enriches the rate of mutant alleles. The difference between Ice-COLD PCR and Full-COLD PCR is the use of a blocking oligonucleotide in Ice-COLDPCR that prevents healthy DNA strands from replicating. In this study, two methods of COLD-PCR and high-resolution melting (HRM) were combined for detection of mutationE542K in exon 9 of PIK3CA and the two methods Full- COLD PCR-HRM and Ice-COLD PCR-HRM are compared.Results: The ice Cold-PCR method, with selective DNA replication, is capable of enriching a small amount of mutated DNA in a mass of wild type DNA. Cell line experiments resulted in almost 4 fold augment in sensitivity by use of ice-COLD PCR/HRM.Conclusion: The results showed us that both methods have advantages and are useful in different applications .Our results clearly demonstrated that ice-COLD PCR/HRM could detect lower levels of mutations in wild-type background. as a sensitive method; therefore, it can prospectively be used in screening of patients with early-stage breast cancers.

کلیدواژه ها:

PIK3CA ، Breast cancer ، COLD-PCR ، Ice COLD-PCR