Analysis and interpretation of mixture DNA using AS-PCR of mtDNA
Analysis and interpretation of mixture DNA using AS-PCR of mtDNA
复制标题
使用线粒体 DNA 的 AS-PCR 分析和解释混合物 DNA
DOI：
10.1002/elps.201800432
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发表时间：
2019-06-01
期刊：
影响因子：
2.9
通讯作者：
Wang, Bao-jie
中科院分区：
文献类型：
--
作者：
Xu, Feng-ling;Ding, Mei;Wang, Bao-jie
采用等位基因特异性引物半巢式PCR和线粒体DNA（mtDNA）测序技术对DNA混合物进行分析和解释，特别是当生物材料降解或含有有限数量的DNA时。SNP-STR标记可用于使用AS-PCR鉴定次要DNA组分;此外，当降解或有限量的DNA混合物不能成功使用SNP-STR标记时，可以使用mtDNA中的SNPs。根据G15043 A、T16362 C和T16519 C三个SNP位点设计了5对等位基因特异性引物。用AS-PCR和测序法获得了线粒体微量组分控制区的序列。将扩增片段的序列与已知可疑物或数据库的序列进行比对和比较。当该测定与T16362 C和T16519 C SNP一起使用时，我们发现它对于检测少量DNA（类似于30 pg）和分析两个贡献者的DNA混合物是高度灵敏的，即使在次要组分和主要组分的比例约为千分之一的情况下。一个例外是基于SNP G15043 A的测试，其需要约300 pg的1% DNA混合物。在模拟的三个贡献者DNA混合物（以1：1：1的比率）中，检测并解释来自每个贡献者的对照区片段。AS-PCR结合半巢式PCR成功地鉴定了各贡献者的mtDNA控制区，为法医学案件中排除嫌疑人提供了生物学证据，尤其是当生物材料降解或DNA含量有限时。
Semi-nested PCR with allele-specific (AS) primers and sequencing of mitochondrial DNA (mtDNA) were performed to analyze and interpret DNA mixtures, especially when biological materials were degraded or contained a limited amount of DNA. SNP-STR markers were available to identify the minor DNA component using AS-PCR; moreover, SNPs in mtDNA could be used when the degraded or limited amounts of DNA mixtures were not successful with SNP-STR markers. Five pairs of allele-specific primers were designed based on three SNPs (G15043A, T16362C, and T16519C). The sequence of mtDNA control region of minor components was obtained using AS-PCR and sequencing. Sequences of the amplification fragments were aligned and compared with the sequences of known suspects or databases. When this assay was used with the T16362C and T16519C SNPs, we found it to be highly sensitive for detecting small amounts of DNA (similar to 30 pg) and analyzing DNA mixtures of two contributors, even at an approximately 1 parts per thousand ratio of minor and major components. An exception was tests based on the SNP G15043A, which required approximately 300 pg of a 1% DNA mixture. In simulated three contributor DNA mixtures (at rate of 1:1:1), control region fragments from each contributor were detected and interpreted. AS-PCR combined with semi-nested PCR was successfully used to identify the mtDNA control region of each contributor, providing biological evidence for excluding suspects in forensic cases, especially when biological materials were degraded or had a limited amount of DNA.