Method development for detection of E545A mutation PIK3CA gene in breast cancer patients using Tm Shift SYBR Green I qPCR

Fuad Al Ahwani(1*), Desriani Desriani(2), Utut Widyastuti(3), Suharsono Suharsono(4)

(1) Depertment of Biotechnology, Bogor Agricultural University, Jl. Raya Darmaga, Bogor, Indonesia
(2) Research center for Biotechnology Indonesian Institute of Science, Jl. Raya Bogor No.507, Cibinong, Indonesia
(3) Depertment of Biotechnology, Bogor Agricultural University, Jl. Raya Darmaga, Bogor, Indonesia
(4) Depertment of Biotechnology, Bogor Agricultural University, Jl. Raya Darmaga, Bogor, Indonesia
(*) Corresponding Author


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 anti-HER2 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 of E545A detection. The developing method used Tm and Ct to identify samples. Based on optimization, the best condition was obtained at optimization 2 at 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 variance coefficient of intra and inter assay (0). Thus, method for E5454A detection mutation was successfully developed.


E545A; breast cancer; Ct; PIK3CA; precision; Tm

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