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目的采用焦磷酸测序技术分析短片段牙釉质蛋白基因进行性别鉴定并用于骨骼及腐败生物检材的检测。方法应用blast软件,确定牙釉质蛋白基因(Amel)上1段含有3个SNP位点及1个插入/缺失(indel)位点的序列作为待测靶序列,设计引物,扩增该段序列,应用焦磷酸测序技术分析扩增序列,进行性别鉴定。对方法进行准确性、灵敏度、种属特异性的测试,并用于对骨骼和高度降解DNA的检测。结果 PCR产物分别为44bp(Amel X)和45bp(Amel Y),女性测序结果为:G/G,T/T,…/…,C/C,男性测序结果为:G/T,T/A,…/C,C/A,分型图谱清晰。应用本文方法检测100份已知性别的DNA样本,结果均正确无误,方法最低DNA模板量为0.5ng,具有较好的人类种属特异性。用于高度降解DNA分析,较IdentifilerTM试剂盒具有更高的成功率且骨骼样本也得到清晰的分型结果。结论本文采用焦磷酸测序技术分析Amel的方法在法医学性别鉴定中有较好的应用价值。
OBJECTIVE: To analyze the sex determination of short-term enamel protein gene by pyrosequencing and to detect bone and spoilage biological samples. Methods Blast software was used to determine the sequence of the first enamel protein gene (Amel) containing three SNP sites and one indel site as the target sequence to be tested. Primers were designed to amplify the sequence, The pyrosequencing technique was used to analyze the amplified sequences for gender identification. Methods are tested for accuracy, sensitivity, and species specificity, and for the detection of skeletal and highly degraded DNA. Results The PCR products were 44 bp (Amel X) and 45 bp (Amel Y), respectively. The sequencing results of female were G / G, T / T, ... / , ... / C, C / A, type map clear. Using this method to detect 100 DNA samples of known sex, the results are correct, the minimum amount of DNA template method 0.5ng, has better human species-specific. For highly degraded DNA analysis, it has a higher success rate than the Identifiler ™ kit and a clear typing result for bone samples. Conclusion The method of pyrosequencing analysis of Amel has good application value in forensic sex identification.