A rapid detection method for single nucleotide polymorphisms based on ligase-agarose gel electrophoresis / 重庆医学

ABSTRACT

Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the ap-peared bands in the agarose gel electrophoresis(AGE).With the 3 SNP sites EGFR,c.2573T>G(L858R),EGFR,c.2582T> A (L861Q)and EGFR,c.2155 G>T(G719C)in epidermal growth factor receptor(EGFR)gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6 cases and 2 cases of heterozygotes mutation in the SNP site of L858R among 62 samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condi-tion.