Liu Jinding, Wang Jiaqi, Zhang Xiaojia, Li Zeqin, Yun Keming, Liu Zhizhen, Zhang Gengqian
School of Forensic Medicine, Shanxi Medicine University, Taiyuan, 030001, Shanxi, China.
The Department of Biochemistry and Molecular Biology, Shanxi Medicine University, Taiyuan, 030001, Shanxi, China.
J Forensic Leg Med. 2017 Feb;46:30-36. doi: 10.1016/j.jflm.2017.01.002. Epub 2017 Jan 17.
Samples containing unbalanced DNA mixtures from individuals often occur in forensic DNA examination and clinical detection. Because of the PCR amplification bias, the minor contributor DNA is often masked by the major contributor DNA when using traditional STR or SNP typing techniques. Here we propose a method based in allele-specific Insertion/Deletion (INDEL) genotyping to detect DNA mixtures in forensic samples. Fourteen INDELs were surveyed in the Chinese Han population of Shanxi Province. The INDELs were amplified using two separate primer-specific reactions by real-time PCR. The difference Ct value of the 2 reactions (D-value) were used for determination of the single source DNA. INDELs types and further confirmed by electrophoresis separation. The minor allele frequency (MAF) was above 0.2 in 10 INDELs. The detection limit was 0.3125 ng-1.25 ng template DNA for real-time PCR in all 14 INDEL markers. For single source 10 ng DNA, the average D-value was 0.31 ± 0.14 for LS type, 6.96 ± 1.05 for LL type and 7.20 ± 1.09 for SS type. For the series of simulated DNA mixture, the Ct value varied between the ranges of single source DNA, depending on their INDEL typing and mixture ratios. This method can detect the specific allele of the minor DNA contributor as little as 1:50 in rs397782455 and rs397696936; 1:100 in rs397832665, rs397822382 and rs397897230; the detection limit of the minor DNA contributor was as little as 1:500-1:1000 in the rest INDEL markers, a much higher sensitivity compared with traditional STR typing. The D-value variation depended on the alternation of dilution ratio and INDEL types. When the dilution was 1:1000, the maximum and minimum D-values were 8.84 ± 0.11 in rs397897230 and 4.27 ± 0.19 in rs397897239 for LL and SS type mixture, the maximum and minimum D-values were 9.32 ± 0.54 in rs397897230 and 4.38 ± 0.26 in rs 397897239 for LL(SS) and LS type mixture, separately. Any D-value between 0.86 and 5.11 in the 14 INDELs indicated the presence of mixture. The separate amplification strategy based on real-time PCR provides a promising and convenient method for detection of unbalanced DNA mixture for Chinese Han population.
在法医DNA检验和临床检测中，经常会出现含有个体不平衡DNA混合物的样本。由于PCR扩增偏倚，在使用传统STR或SNP分型技术时，次要贡献者的DNA往往会被主要贡献者的DNA所掩盖。在此，我们提出一种基于等位基因特异性插入/缺失（INDEL）基因分型的方法，用于检测法医样本中的DNA混合物。我们对山西省汉族人群中的14个INDEL进行了调查。通过实时PCR使用两个单独的引物特异性反应扩增INDEL。将这两个反应的差异Ct值（D值）用于确定单源DNA。通过电泳分离对INDEL类型进行进一步确认。10个INDEL的次要等位基因频率（MAF）高于0.2。在所有14个INDEL标记中，实时PCR的检测限为0.3125 ng - 1.25 ng模板DNA。对于单源10 ng DNA，LS型的平均D值为0.31±0.14，LL型为6.96±1.05，SS型为7.20±1.09。对于一系列模拟DNA混合物，Ct值根据其INDEL分型和混合比例在单源DNA的范围内变化。该方法在rs397782455和rs397696936中可检测到低至1:50的次要DNA贡献者的特定等位基因；在rs397832665、rs397822382和rs397897230中为1:100；在其余INDEL标记中，次要DNA贡献者的检测限低至1:500 - 1:1000，与传统STR分型相比灵敏度更高。D值变化取决于稀释比例和INDEL类型的变化。当稀释比例为1:1000时，对于LL和SS型混合物，rs397897230中的最大和最小D值分别为8.84±0.11和4.27±0.19；对于LL(SS)和LS型混合物，rs397897230中的最大和最小D值分别为9.32±0.54和4.38±0.26。14个INDEL中任何介于0.86和5.11之间的D值都表明存在混合物。基于实时PCR的单独扩增策略为检测汉族人群不平衡DNA混合物提供了一种有前景且便捷的方法。
