Validation Studies on the Forensic Analysis of Restriction Fragment Length Polymorphism (RFLP) on LE Agarose Gels Without Ethidium Bromide: Effects of Contaminants, Sunlight, and the Electrophoresis of Varying Quantities of Deoxyribonucleic Acid (DNA)

Abstract

This study was designed to analyze the effects of sunlight, various contaminants (those found typically in forensic samples) and the electrophoresis of varying quantities of DNA on the restriction fragment length polymorphism (RFLP) patterns produced from DNA isolated from blood and semen stains. The DNA RFLP patterns were obtained following Hae III restriction enzyme digestion, low electroendosmotic (LE) agarose gel electrophoresis (in the absence of ethidium bromide), Southern transfer, hybridization with DNA probes detecting highly polymorphic variable number of tandem repeats (VNTRs) and autoradiography. Computer assisted image analyses were used to detect variations in RFLP band sizes in relation to control samples. Comparisons between the samples were made for the presence of high molecular weight DNA, the ability to achieve a complete restriction digestion, and the RFLP fragment sizes obtained. The results demonstrate that high molecular weight DNA can be obtained when blood and semen stains are subjected to environmental and contaminating factors. The RFLP allele sizes were not significantly affected by environmental conditions, contamination factors or by loading varying amounts of DNA. This study serves to further document the reliability and validity of DNA typing for forensic applications.