Nicklas Janice A, Noreault-Conti Trisha, Buel Eric
Vermont Forensic Laboratory, Department of Public Safety, Waterbury, VT 05671, USA.
J Forensic Sci. 2012 Mar;57(2):466-71. doi: 10.1111/j.1556-4029.2011.02001.x. Epub 2011 Dec 8.
Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real-time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples.
了解证据性DNA样本的降解状态将有助于选择合适的分析方法（标准短串联重复序列[STRs]、微型STRs或线粒体DNA[mtDNA]）。本文描述了一种Plexor®技术/实时PCR DNA降解检测方法的开发，该方法使用一个通用正向引物和两个反向引物（不同荧光团）来生成两个Alu扩增子（63和246碱基对）。这种非常灵敏的检测方法针对反应体积、循环数、退火/延伸时间和温度进行了优化。使用经DNA酶I降解的DNA样本，短扩增子浓度与长扩增子浓度的比值（降解率）随降解时间增加。对各种环境降解样本（老化、阳光、高温）以及七种常见法医抑制剂进行了实验。发现降解率可预测在分析受损样本时观察到的较大STR基因座的丢失情况。
