Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, 11 Frederik V's Vej, DK-2100 Copenhagen, Denmark.
Forensic Sci Int Genet. 2009 Dec;4(1):34-42. doi: 10.1016/j.fsigen.2009.04.004. Epub 2009 May 7.
A multiplex assay with 49 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was validated for forensic genetic casework and accredited according to the ISO 17025 standard. The multiplex assay was based on the SNPforID 52plex SNP assay [J.J. Sanchez, C. Phillips, C. Børsting, K. Balogh, M. Bogus, M. Fondevila, C.D. Harrison, E. Musgrave-Brown, A. Salas, D. Syndercombe-Court, P.M. Schneider, A. Carracedo, N. Morling, A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (2006) 1713-1724], where 52 fragments were amplified in one PCR reaction. The SNPs were analysed by single base extension (SBE) and capillary electrophoresis. Twenty-three of the original SBE primers were altered to improve the overall robustness of the assay and to simplify the analysis of the SBE results. A total of 216 samples from 50 paternity cases and 33 twin cases were typed at least twice for the 49 SNPs. All electropherograms were analysed independently by two expert analysts prior to approval. Based on these results, detailed guidelines for analysis of the SBE products were developed. With these guidelines, the peak height ratio of a heterozygous allele call or the signal to noise ratio of a homozygous allele call is compared with previously obtained ratios. A laboratory protocol for analysis of SBE products was developed where allele calls with unusual ratios were highlighted to facilitate the analysis of difficult allele calls. The guidelines for allele calling proved to be highly efficient for the detection of DNA mixtures and contaminated DNA preparations. DNA from two individuals was mixed in seven different ratios ranging from 1:1 to 1:10; all mixtures were easily identified as mixtures.
一种包含 49 个常染色体单核苷酸多态性（SNP）的多重分析方法被开发出来，用于人类识别，该方法经过了法医遗传学案件的验证，并根据 ISO 17025 标准进行了认证。该多重分析方法基于 SNPforID 52plex SNP 分析[J.J. Sanchez, C. Phillips, C. Børsting, K. Balogh, M. Bogus, M. Fondevila, C.D. Harrison, E. Musgrave-Brown, A. Salas, D. Syndercombe-Court, P.M. Schneider, A. Carracedo, N. Morling, A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (2006) 1713-1724]，其中 52 个片段在一个 PCR 反应中被扩增。SNP 通过单碱基延伸（SBE）和毛细管电泳进行分析。23 个原始 SBE 引物被改变，以提高分析方法的整体稳健性，并简化 SBE 结果的分析。对来自 50 个亲子案例和 33 个双胞胎案例的 216 个样本进行了至少两次 49 个 SNP 的分型。在批准之前，由两名专家分析员独立分析所有电泳图谱。基于这些结果，制定了详细的 SBE 产物分析指南。根据这些指南，杂合子等位基因调用的峰高比或纯合子等位基因调用的信号噪声比与以前获得的比值进行比较。开发了一种用于 SBE 产物分析的实验室方案，其中突出显示具有不寻常比值的等位基因调用，以方便困难等位基因调用的分析。等位基因调用指南被证明对检测 DNA 混合物和污染 DNA 制剂非常有效。将两个人的 DNA 以 1:1 到 1:10 的七种不同比例混合；所有混合物都很容易被识别为混合物。
