3种DNA提取法在污染严重混合斑分型中的应用比较

Comparison of three DNA extraction methods for genotyping of difficulty mixed stains

目的比较Chelex-100法、酚/氯仿法和二氧化硅膜法3种DNA提取法在污染严重混合斑分型中的应用效果。方法从日常案例中收集污染严重的混合斑25份,差异消化法分离精子后同时用Chelex-100法、酚/氯仿法和二氧化硅膜技术3种方法提取DNA,采用PCR-STR技术对D19S253、FGA和CSF1PO 3个基因座进行分型,Gel-Pro软件处理电泳图谱,SPSS软件分析比较不同方法之间的差异。结果采用Chelex-100法提取DNA,25份检材分型结果均未成功;采用酚/氯仿法,25份检材中10份分型成功,3份检材FGA和CSF1PO基因座可分型,4份检材CSF1PO基因座可分型;采二氧化硅膜纯化法,25份检材均成功分型;酚/氯仿法和二氧化硅膜法两种方法比较,结果存在显著性差异(P<0.05)。结论二氧化硅膜纯化技术可以有效去除PCR抑制物,提取的DNA扩增效果明显优于Chelex-100法和酚/氯仿法,具有较高的应用价值。

Objective To Compare three DNA extraction methods for genotyping of difficulty mixed stains. Methods 25 difficulty mixed stain samples were collected. After separated from vaginal epithelial cell by differential digestion method, sperm DNA was isolated using Chelex-100, phenolic-chloroform and silica purification methods respectively. Three STR loci, D19S253, FGA and CSF1PO, were typed by polymerase chain reaction and polyacrylamide gel electrophoresis techniques, and the bands of different methods were analyzed and compared with Gel-Pro and SPSS software. Results With Chelex-100 method, no amplification product in all three loci was detected for all samples. With phenol-chloroform method, 10 samples were genotyped successfully in all three loci, 3 sample in FGA and CSF1PO, and 4 samples in CSF1PO locus. When silica membrane technique was used, however, all 3 loci for all samples were typed successfully. The band density was different significantly between phenolic chloroform and silica purification methods （ P 〈 0.05 ）. Conclusion The results demonstrated that silica purification technique was more effective in eliminating PCR inhibitors than Chelex-100 and phenol-chloroform methods, and can be used to DNA extraction of contaminative biological samples in forensic practice.