生物检材DNA碱性裂解提取法的优化与改良

Optimization and Improvement of the Alkaline Lysis Method for the extraction of DNA from Forensic Biological Samples

目的通过对DNA碱性裂解提取法进行优化和改进,建立一种操作更简便、检验快速、结果更可靠的生物检材DNA提取方法。方法以抗凝血作为检验样本,通过改变提取试剂的浓度、pH值、保存时间及孵化样本的温度、时间等条件,观测对检验结果峰值的影响,以确定提取试剂最佳条件,DNA提取物最佳保存条件。通过用改良后的碱性裂解法及Chelex100法同时提取不同量的抗凝血样本、各类法医生物检材,用不同厂家的DNA试剂盒扩增,对碱性裂解法的灵敏度、适应性和适用性进行评估。结果改良后的碱性裂解法只需将生物检材用NaOH(0.25mol/L)99℃孵化8min,振荡后加入TrisHCl(0.05mol/L,pH=6.5)中和液,离心后直接扩增检测。DNA提取物于-20℃冰箱可长期保存。对于毛发及精斑类检材优于Chelex100法。DNA提取物适用于现有各种DNA试剂盒扩增检测,其灵敏度与Chelex100法相似。结论改良后的碱性裂解法,操作简便、检验快速、结果可靠,可适合于法庭科学的检案与建库。

Objective To establish a simple,fast and reliable method for extracting DNA from forensically relevant specimens through optimization and improvement of the alkaline lysis protocol.Methods With the anticoagulated blood as test samples,the optimal conditions of the extraction reagents were determined according to the influence on the results of DNA typing of different conditions,including different reagent concentration,pH value and storage time,and different incubation temperature and time.Meanwhile,the optimal preservation conditions of the extracts were also determined by comparing DNA typing results of the extracts stored at different temperature.In addition,compared with the chelex 100 protocol,the sensitivity,adaptability and applicability of the modified alkaline lysis method were evaluated by using different quantity of blood,different types of forensically relevant specimens,different DNA typing kits.Results The improved alkaline lysis protocol summarized as follows： NaOH（0.25mol/L） was used to incubate the forensic biological samples at 99℃ for 8 min,then added the neutralizing liquid,TrisHCl（0.05mol/L,pH=6.5）.After vortex and centrifuge,the supernatant was used for amplification.The genomic DNA extracted with this method could be stored at-20℃ for long-term preservation.This method could extract DNA from all kinds of forensic biological samples,and for hair and seminal stains,it was better than the chelex 100 method.The extracts with this method were applicable to a variety of DNA amplification kit,and the sensitivity was similar to that of the Chelex 100 method.Conclusion The improved alkaline lysis protocol is a simple,fast and reliable method for the extraction of DNA from forensic biological samples,and can be used to forensic practice and the DNA database construction.