School of Forensic Medicine, China Medical University, PR China; Department of Forensic Medicine, Baotou Medical College, PR China.
Forensic Sci Int Genet. 2014 Jan;8(1):143-6. doi: 10.1016/j.fsigen.2013.08.013. Epub 2013 Sep 7.
Several commercial multiplex PCR kits for the amplification of short tandem repeat (STR) loci have been extensively applied in forensic genetics. Consequently, large numbers of samples have been genotyped, and the number of discordant genotypes observed has also increased. We observed allele dropout with two novel alleles at the STR loci TH01 and D13S317 during paternity testing using the AmpFℓSTR Identifiler PCR Amplification Kit. The lost alleles reappeared when alternative PCR primer pairs were used. A sequence analysis revealed a G-to-A substitution 82 bases downstream of the last TCAT motif of the repeat region at the TH01 locus (GenBank accession: D00269) and a G-to-T substitution 90 bases upstream of the first TATC motif of the repeat region at the D13S317 locus (GenBank accession: G09017). The frequencies of these two point mutations were subsequently investigated in the Chinese population using sequence-specific primer PCR (SSP-PCR), but neither of these mutations was detected in any of the samples tested. In addition, the DNA samples in which the mutations were identified were amplified to type the point mutations by SSP-PCR to determine the corresponding STR alleles at the two loci. Subsequently, the amplified PCR products with different point mutations and STR repeat numbers were directly sequenced because this strategy overcomes the appearance overlapping peaks generated by different STR alleles and accurately characterizes genotypes. Thus, our findings not only provide useful information for DNA databases and forensic identification but also establish an effective strategy for typing STR alleles with primer binding site mutations.
已有多个商业化的短串联重复序列（STR）基因座多重 PCR 试剂盒被广泛应用于法医遗传学。因此，大量的样本被进行了基因分型，并且观察到的不一致基因型的数量也有所增加。在使用 AmpFℓSTR Identifiler PCR 扩增试剂盒进行亲子鉴定时，我们观察到在 STR 基因座 TH01 和 D13S317 上有两个新等位基因出现等位基因丢失。当使用替代的 PCR 引物对时，丢失的等位基因重新出现。序列分析显示，在 TH01 基因座重复区最后一个 TCAT 基序下游 82 个碱基处（GenBank 登录号：D00269）发生了 G 到 A 的取代，在 D13S317 基因座重复区第一个 TATC 基序上游 90 个碱基处发生了 G 到 T 的取代（GenBank 登录号：G09017）。随后，我们使用序列特异性引物 PCR（SSP-PCR）在中国人群中调查了这两个点突变的频率，但在测试的任何样本中都没有检测到这些突变。此外，还对鉴定出突变的 DNA 样本进行扩增，通过 SSP-PCR 对这些点突变进行分型，以确定这两个基因座的相应 STR 等位基因。随后，我们直接对具有不同点突变和 STR 重复数的扩增 PCR 产物进行测序，因为这种策略克服了由不同 STR 等位基因产生的峰重叠现象，并准确地对基因型进行了特征描述。因此，我们的发现不仅为 DNA 数据库和法医鉴定提供了有用的信息，而且还建立了一种有效的策略，用于对引物结合位点突变的 STR 等位基因进行分型。
