Institute of Forensic Medicine, Medical University of Gdańsk, Dębowa 23, 80-204, Gdańsk, Poland.
Int J Legal Med. 2013 Mar;127(2):309-19. doi: 10.1007/s00414-012-0764-9. Epub 2012 Sep 1.
Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).
降解的 DNA 经常在法医遗传学实验室中进行分析。可靠的降解 DNA 分析非常重要,因为其结果会影响专家证言的质量和可靠性。最近,已经提出了许多全基因组扩增 (WGA) 方法作为预扩增工具。它们的工作前提是能够从单个细胞的 DNA 量中产生微克量的 DNA。我们选择、研究并比较了七种 WGA 方法,以评估它们“恢复”降解和非降解 DNA 的能力:简并寡核苷酸引物 PCR、引物延伸预扩增 PCR、GenomePlex™ WGA 商业试剂盒 (Sigma)、多重置换扩增、GenomiPhi™ 扩增试剂盒 (Amersham Biosciences)、限制和环化辅助滚环扩增以及钝端连接介导的 WGA。使用 SGMPlus、YFiler、mtDNA 和 Y 染色体 SNP 分型分析和比较了这些方法的效率和可靠性。对于非降解 DNA,GenomiPhi 和 PEP 方法的结果最好。对于降解的 DNA(200 bp),GenomePlex 获得了最佳结果,其成功地扩增了严重降解的 DNA(100 bp),从而能够正确地对 mtDNA 和 Y-SNP 基因座进行分型。WGA 在法医遗传学中分析低拷贝数 DNA 或降解的 DNA 时可能非常有用,尤其是在引入一些改进措施(样本混合和重复 DNA 分型)之后。