用分子克隆技术构建D12S391基因座等位基因分型标准物及群体遗传学研究

目的 解决长期困扰短串联重复序列（short tandem repeat,STR）分型上存在的准确性和标准化问题。方法 先用PCR扩增出D12S391基因座的9个等位基因片段，将其插入pUC重组质粒中，经DNA测序分析证实插入片段的结构及大小，用国际标准将插入的等位基因片段进行命名，最后经转染、扩大培养、扩增及再鉴定后，制备出标准的D12S391等位基因分型标准物。结果 应用此法制备出大量的D12S391基因座等位基因分型标准物，并将其用于调查该基因座在德国Mainz地区、日本Miyazaki地区及中国成都汉族、北京汉族、新疆维吾尔族和甘肃回族6个群体中的基因型分布频率。D12S391基因座在各群体中均有较高的多态性，其非父排除概念及个人识别能力分别为0.609-0.786和0.940-0.952。结论 该法制备的STR基因座等位基因分型标准物在法医科学实践中应用价值极高，D12S391基因座是一个非常适合于群体遗传学研究和法医科学应用的遗传标记。

Study on the construction of standard D12S391 allelic ladder and its genetic polymorphism in six populations

OBJECTIVE: To resolve the problem of the accuracy and standardization of short tandem repeat-polymerase chain reaction (STR-PCR) typing in forensic practice, the authors have designed a new method of producing standard D12S391 allelic ladder. METHODS: Nine different PCR amplified D12S391 allelic fragments were isolated from the gel, eluted into the distilled water and re-amplified by PCR. The purified allelic fragments were then blunt-end subcloned individually into the pUC plasmid vectors and transfected into competent E.coli DH5 alpha(TM) cells. The sequencing results confirmed that the size and the structure of the inserts were correct. The recombinant plasmids DNA with 9 inserts were then used as templates for PCR re-amplification to generate D12S391 standard ladder. RESULTS: With the ladder, the authors studied the genetic polymorphisms of D12S391 locus in six populations (German, Japanese and Chinese south-western Han, northern Han, Weiwu'er and Hui populations), and the respective primary data in the six populations were obtained. D12S391 locus showed high polymorphism in all six populations, and its exclusion power and discrimination power are 0.609-0.786 and 0.940-0.952 respectively. CONCLUSION: The results demonstrate that the standard ladder generated via this method is excellent, and D12S391 locus is robust for genetic research and forensic application.