بررسي و مقايسه امكان تشخيص جهش ژن PIK3CA توسط روش هاي COLD-HRM PCR و PCR-Sequencing در رقت هاي مختلف DNA جهش يافته

Assess and Compare the PIK3CA Gene Mutation Detection by PCR-Sequencing and Full COLD-HRM PCR Methods in Different Dilutions of DNA Mutations

Background Purpose: PIK3CA mutations appear to have a clinical significance in breast cancer, So that a third of breast cancers are caused by somatic mutations in the PIK3CA gene. Therefore PIK3CA mutations are biomarkers for breast cancer prognosis and diagnosis. H1047R mutation in PIK3CA gene account approximately %55 of mutations reported for this gene, according to COSMIC Database. Molecular profiling of somatic mutations in cancer often requires the identification of low-level DNA mutations within an excess of wild-type DNA. However traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer. COLD-PCR (CO-amplification at Lower Denaturation Temperature) resolves several limitations in low-level mutation detection by using critical denaturation temperatures to enrich mutant-containing amplicons during PCR. on the other hand High-resolution melting analysis (HRM) is a highly sensitive, robust, rapid, cost-effective mutation analysis technique and powerful tool for screening and detecting genomic mutations efficiently. In this study, we designed a Improved protocol to detect mutation of PIK3CA gene based on COLD-PCR and HRM. Materials and Methods: Serial dilutions of PIK3CA mutated DNA from the BT-20cell lines in wild-type DNA of MCF-7 cell line were prepared in order to asses method sensitivity. Dilutions were amplified via PCR-Sequencing and full COLD-HRM PCR for H1047R mutation in PIK3CA gene and the 2 approaches were compared. Results: Dilution experiments indicated an approximate 15 fold improvement in selectivity with COLD-HRM PCR. PCR-sequencing exhibited mutation-detection approximately 12.5% whereas COLD-HRM PCR exhibited approximately 0.8% mutant-to-wild-type ratio. This method is a powerful method to evidence the presence of low-level mutation and significantly improved detection of low-level mutant alleles in background of wild-type. Conclusion: In this study, H1047R mutations were investigated by using COLD-HRM PCR. In this study illustrates that the correct identification of less-represented mutations in background of wild-type can be significantly improved with COLD-PCR combined with HRM, without requiring expensive and time consuming procedures and while maintaining a closed-tube approach. Keywords: Breast cancer, PIK3CA gene, H1047R mutation, COLD-HRM PCR

-Background Purpose: PIK3CA mutations appear to have a clinical significance in breast cancer, So that a third of breast cancers are caused by somatic mutations in the PIK3CA gene. Therefore PIK3CA mutations are biomarkers for breast cancer prognosis and diagnosis. H1047R mutation in PIK3CA gene account approximately %55 of mutations reported for this gene, according to COSMIC Database. Molecular profiling of somatic mutations in cancer often requires the identification of low-level DNA mutations within an excess of wild-type DNA. However traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer. COLD-PCR (CO-amplification at Lower Denaturation Temperature) resolves several limitations in low-level mutation detection by using critical denaturation temperatures to enrich mutant-containing amplicons during PCR. on the other hand High-resolution melting analysis (HRM) is a highly sensitive, robust, rapid, cost-effective mutation analysis technique and powerful tool for screening and detecting genomic mutations efficiently. In this study, we designed a Improved protocol to detect mutation of PIK3CA gene based on COLD-PCR and HRM. Materials and Methods: Serial dilutions of PIK3CA mutated DNA from the BT-20cell lines in wild-type DNA of MCF-7 cell line were prepared in order to asses method sensitivity. Dilutions were amplified via PCR-Sequencing and full COLD-HRM PCR for H1047R mutation in PIK3CA gene and the 2 approaches were compared. Results: Dilution experiments indicated an approximate 15 fold improvement in selectivity with COLD-HRM PCR. PCR-sequencing exhibited mutation-detection approximately 12.5% whereas COLD-HRM PCR exhibited approximately 0.8% mutant-to-wild-type ratio. This method is a powerful method to evidence the presence of low-level mutation and significantly improved detection of low-level mutant alleles in background of wild-type. Conclusion: In this study, H1047R mutations were investigated by using COLD-HRM PCR. In this study illustrates that the correct identification of less-represented mutations in background of wild-type can be significantly improved with COLD-PCR combined with HRM, without requiring expensive and time consuming procedures and while maintaining a closed-tube approach. Keywords: Breast cancer, PIK3CA gene, H1047R mutation, COLD-HRM PCR