关键词: D6S1043基因座, STR, 质粒DNA标准物质

Abstract: 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.

Key words: D6S1043 locus, STR, plasmid DNA reference material