Marino M A, Weaver K R, Tully L A, Girard J E, Belgrader P
Developmental Laboratory, Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, Washington DC, USA.
Electrophoresis. 1996 Sep;17(9):1499-504. doi: 10.1002/elps.1150170915.
Polymerase chain reaction (PCR)-based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low-stringency sequence-specific PCR (LSSP-PCR) is an example of a recent method that does not require additional biochemical treatments. The analysis of LSSP-PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D-loop region is described. The DNA from five individuals were amplified (first step) using sequence-specific primers to produce 1021 bp fragments containing the D-loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO-1 and laser-induced fluorescence (LIF) detection. The LSSP-PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.
基于聚合酶链反应（PCR）的DNA分型技术在法医学中常用于身份鉴定。那些区分单核苷酸多态性（SNP）的检测方法，除了PCR之外还需要其他生化反应来识别序列多态性。低严谨度序列特异性PCR（LSSP-PCR）是一种最近出现的不需要额外生化处理的方法。本文描述了通过毛细管电泳（CE）分析LSSP-PCR以区分高度多态的线粒体DNA（mtDNA）D环区域。使用序列特异性引物对来自五个人的DNA进行扩增（第一步），以产生包含D环区域的1021 bp片段。通过使用CE和紫外检测的电洗脱法分离每个片段，并使用在低严谨度条件下退火的单个引物进行第二次扩增（第二步）。这为每个样品生成了一系列PCR产物或图谱，通过使用嵌入剂TOTO-1和激光诱导荧光（LIF）检测的CE对其进行解析和分析。每个个体的LSSP-PCR图谱都是独特的，表明该技术可能适用于法医学身份鉴定。
