应用富含GC区SNP分型的序列特异性PCR新方法

Objective: To develop a sequence-specific polymerase chain reaction (SS-PCR) method for detection of single nucleotide polymorphism (SNP) in GC rich region and analyze the C729T SNP in the exon 3 of estrogen receptor α gene of endometrial cancer (EC). Methods: We selected 22 EC and 42 controls. A new GC rich region SS-PCR was developed. The two internal specific primers, identical to the two single strands of double strand DNA, were designed, and the 3' end of the primer coincided with the SNP locus. The PCR extension was controlled by the 3' end primer and the SNP was determined according to the bands of the extension product. The specificity of the extension reaction was increased by introducing a mismatched base at the third position upstream in the 3' end of the SNP specific primer. This method was used to confirm C729T genotypes in the exon 3 SNP, followed by direct sequencing. Results: The results showed that the SS-PCR was appropriate for genotyping C729T SNP of exon 3 in human ERα gene. Conclusion: A GC rich region SS-PCR method developed can be successfully applied in C729T SNP determination in the exon 3 of estrogen receptor α gene of endometrial cancer.