Detection of survivin gene methylation with SYBR Green Ⅰ real-time fluorescent PCR / 国际检验医学杂志

ABSTRACT

Objective To establish a real-time PCR based method for detection of survivin gene samples methylation with dye SYBR Green Ⅰ.Methods DNA samples from 25 pairs of gastric cancer tissue and matched normal gastric tissues were digested with mCpG-sensitive Hpa Ⅱand Msp Ⅰ, and detected by SYBR Green Ⅰ real-time PCR for survivin exon 1 specific primers. The concentration of digested DNA was monitored by real-time quantitative PCR with survivin intron 2 specific primers. A normal genomic DNA which served as a standard was diluted serially 10-fold to determine the sensitivity of real-time PCR. The products were further verified by agarose gel electrophoresis analysis.Results After HpaⅡ enzyme digestion, a peak of PCR product derived from methylated targeted gene was found in the melting curve at Tm (91.5±0.5) ℃, which was verified as 338 bp strap. The control gene (survivin intron 2) characteristic of Tm (79.5±0.5) ℃ was amplified in all digested samples, indicating no non-specific degradation occurred in the genome DNA. The sensitivity of this method was 100 copies/μL. The new established PCR system was applied to detecting 25 cases of lung cancer tissue samples. As a result, the frequency of survivin exon 1 demethylation was 96%.Conclusion As a reliable new method for detection of survivin exon 1 methylation, SYBR Green Ⅰ real-time fluorescent PCR assay is rapid, accurate, sensitive, timing and simple.