Chelex-100提取生物检材DNA实时PCR定量研究

Real time PCR quantificational study of DNA extracted by Chelex-100 method

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。

Objective To study the relation between the quantity of DNA extracted by Chelex-100 method and multiplex STRs analysis. Methods DNA extracted from a variety of common forensic casework specimens were quantified by using Real-time PCR, and then amplified with AmpFLSTR Identifiler^TM PCR Amplification kit. Results According to the results of quantification, the quantities of DNA extracted from 113 samples by Chelex-100 method were adjusted to 0.5 - 3ng for establishing 8μl amplification system, and in this condition, most of 113 forensic casework specimens could be successfully genotyped. Conclusion When the quantity of DNA extracted by Chelex-100 method ranged from 0.5ng to 3ng, most results of multiplex STRs analysis were satisfying. Moreover, the amplification effect of 1μl DNA template was better than 3μl DNA template when the concentrations of extracted DNA were more than 0.5ng/μl.