Method Development for Detection of E545A Mutation PIK3CA Gene in Breast Cancer Patients Using Tm Shift SYBR Green I qPCR

Abstract

E545A is one of the point mutations, and its frequency is high in PIK3CA gene (3.8%), particularly breast cancer patients in Singapore (13.8%) and Mexico (11.5%). In addition to induce breast cancer, the mutation also caused resistance of antiHER2 in HER2 cancer subtype. The tremendous effect of this mutation was not supported by affordable detection method. This study aimed to develop a feasible and sensitive method for E545A detection. The developing method used Tm and Ct to identify samples. Based on optimization, the best condition was obtained with annealing temperature of 65°C. At this condition, Tm and Ct of each sample were (a) exon 9 (78.4°C and 13.005±0.007) and (b) E5454A (80.4°C and 10.07±0.1). This method also demonstrated good precision as observed in coefficient variance of intra and inter assay (0). Thus, method for E5454A detection mutation was successfully developed.

The sensitivity and efficacy method of PIK3CA exon 9 E545A as a high diagnostic accuracy in breast cancer

“…The best primer proportions for PIK3CA E545A detection are 0.03 μM (long FW): 0.00175 μM and (short FW): 0.03 μM. This proportion formulation is better than that reported in a previous study [18]. In this experiment, we increased the primer annealing optimization to above 65 °C, namely 65 °C, 66.2 °C, 67.5 °C, and 68 °C, in order to increase the specificity primer to recognize the target.…”

“…Both primer sets have the same forward primer that recognized WT, which is 5′-GCGGGCtcctctctctgaaatcactga-3′, and the same reverse primer, which is 5′-gggccggatccattttagcacttacctgtg-3′. The first forward primer for the first primer set was 5′-GCGGGCAGGGCGGCtcctctctctgaaatcaccgc-3′, which was the same as that reported in a previous study [18]. Meanwhile, the second primer set was 5′-GCGGGCAGGGCGGCtcctctctctgaaatcattgc.…”

“…Furthermore, the removal of the extension step could also be considered for increasing the primer specificity. The presence of the extension step in E545A development methods will decrease primer specificity [18].…”

“…In a previous report, we successfully developed a PIK3CA E545A detection method using the Tm Shift SYBR Green I approach [18]. Tm Shift SYBR Green I has been described as a low-cost, simple, and massive detection method.…”