人D6S1043-1型STR质粒DNA标准物质的研制

短串联重复序列（short tandem repeat，STR）是存在于人类基因组中的一类具有长度多态性的DNA序列，由含2~6个碱基对的重复单位串联构成。DNA STR分型检验是当前法庭科学进行个体识别和亲缘鉴定的主要依据。STR分型标准物质是DNA STR检验量值溯源的基础和关键物质，对其进行研究将极大推动我国法庭科学DNA STR检验的标准化进程。以STR基因座D6S1043为例，介绍人类基因组中STR序列的质粒DNA标准物质的制备过程。首先，合成包含13个重复单元（\ATCT\]13）的D6S1043序列（定义为D6S1043-1）并将其与pUC57载体连接构建重组质粒，制备质粒溶液。其次，通过酶切鉴定、Sanger测序、多种STR分型试剂盒的分型鉴定等方法检验DNA序列的准确性。再次，采用微滴式数字PCR方法（droplet digital PCR，ddPCR）检测质粒浓度，评估质粒溶液的均匀性和稳定性。最后，由8家实验室协作，测定浓度标准值，综合分析均匀性检验、稳定性检验、定值过程等引入的不确定度，得到总不确定度；由6家实验室协作，测定D6S1043-1的分型值。结果表明：该标准物质的均匀性、稳定性良好，在-20 ℃可保存6个月，在4 ℃可保存14 d；核酸拷贝数为（7.7±1.2）×103 copies·μL-1；在D6S1043基因座上的分型值为13。人D6S1043-1型STR质粒DNA标准物质 \编号：GBW（E）091072\] 是我国法庭科学DNA检验领域首批有证标准物质之一，其研制过程为其他STR基因座的质粒DNA标准物质的研制提供了一定的借鉴。

Preparation of Plasmid DNA Reference Material Containing Human STR D6S1043-1 Fragment

Short tandem repeat (STR) is a kind of DNA sequence with length polymorphism existing in human genome, which consists of 2~6 base pairs of repeating units connected in series. DNA STR typing test is the main basis for individual identification and genetic identification in forensic science at present. STR typing reference materials are the basis and key materials for the traceability of DNA STR test, and the research on them will greatly promote the standardization process of forensic DNA STR test in China. Taking STR locus D6S1043 as an example, the preparation process of plasmid DNA reference materials for STR sequences from human genome was introduced. At first, a DNA sequence (defined as D6S1043-1) containing 13 repeat units (\ATCT\]13) of the D6S1043 locus was synthesized and ligated to pUC57 vector to construct a recombinant plasmid, thus preparing a plasmid solution. Secondly, the accuracy of DNA sequence was tested by enzyme digestion identification, Sanger sequencing, and typing identification of multiple STR typing kits. Thirdly, the concentration of plasmid was detected by using droplet digital PCR (ddPCR) method, as well as the uniformity and stability of plasmid solution. Finally, the concentration standard value was determined through the cooperation of 8 laboratories, and the total uncertainty was obtained through comprehensively calculating the three kinds of uncertainties introduced by the homogeneity test, stability test and the determination process. And the typing value of D6S1043-1 was confirmed by typing identification of multiple STR typing kits in 6 laboratories. The results showed that the reference material had good uniformity and stability, and could be stored for 6 months at -20 ℃ and 14 d at 4 ℃; the concentration of the plasmid was (7.7±1.2)×103 copies·μL-1; the typing value was 13 at the D6S1043 locus. Human D6S1043-1 STR plasmid DNA reference material \code: GBW(E)091072\] had become one of the first certified reference materials in the field of forensic DNA test, whose preparation process provided a certain reference for the preparation of plasmid DNA reference materials for other STR loci.