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A method was developed for genotyping of single nucleotide polymorphism (SNP) by real-time polymerase chain reaction (PCR) coupled with high specific invader assay. The concentrations of flap endonuclease 1 (FEN1 enzyme) and wild type detection probe were optimized to reduce the background signal. Under the optimal conditions including 1×buffer, 250 μM dNTPs, 0.5 μM each PCR primer, 0.05 μM invasive oligo probe, 0.125 μM wild detection probe, 0.5 μM mutant detection probe, 0.25 μM each fluorescence resonance energy transfer (FRET) probe, 1 U Taq DNA polymerase, 1.5 U FEN1, and approximate 40-200 ng of genomic DNA, the background signals of wild-type sample and mutant-type sample were dramatically decreased and the background interference to the esults was thus eliminated. The reaction program was initial denaturation at 95 °C for 3 min, followed by 10 cycles (95 °C for 20 s, 67 °C for 60 s, 70 °C for 30 s) and 35 cycles (95 °C for 20 s, 63 °C for 60 s, read the fluorescence signal, 70 °C for 30 s). In the experiment, a total of 21 cases of ALDH2*2, 19 cases of CYP2C19*2 and 19 cases of CYP2C19*3 were analyzed by the established method, and the results showed that the genotypes of ALDH2*2 were 10 cases of GG homozygote, 8 cases of GA heterozygote and 3 cases of AA homozygote; the genotypes of CYP2C19*2 were 9 cases of GG homozygote, 8 cases of GA heterozygote and 2 cases of AA homozygote; and the genotypes of CYP2C19*3 were 18 cases of GG homozygote and 1 case of GA heterozygote. These results were consistent with the detection results by pyrosequencing. The established method was specific, simple, short time-consuming and low cost, and could be used for SNP genotyping with no-pollution in single tube. |
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