焦磷酸测序分析短片段牙釉质蛋白基因进行性别鉴定

Gender identification by pyrosequencing of short fragments of amelogenin gene

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 establish a pyrosequencing method for detecting a short amelogenin fragment for gender identification, and evaluate its application value for samples of bone and highly-degraded DNA. Methods The DNA sequence consisting of 3 SNPs and one indel between AMELX and AMELY, was selected as target sequence by using blast, and amplified with selfdesigned primers. The amplification products were sequenced by the pyrosequencing method to identify the gender of samples. The method was also performed to evaluate its reliability, sensitivity, species-specificity and the capacity to analyze bones and highly-degraded DNA samples. Results The PCR products were 44 bp pyrosequencing results of female samples were G/G, and 45 bp for AMELX and AMELY respectively. The T/T, del/del, C/C, whereas the sequence of male samples were G/T, T/A, del/C, C/A. 100 randomly chosen DNA samples already known the gender got clear pyrographs and correct testing results. The sensitivity of the technique was 0. 5 ng template DNA. No specific peak was found in any detected animals or organisms except in monkeys. For highly-degraded DNA samples, this method obtained more accurate results than the IdentifilerTMkit did. Moreover, 4 bone samples obtained clear pyrograph by the method. Condusion The new method based on pyrosequencing of short PCR products of the amelogenin gene has good application value in forensic gender identification.