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.
This study describes a method for establishing match criteria used in forensic DNA typing. The validity of applying different match criteria based upon the molecular weight of a DNA band is discussed. The match criteria presented allow visually matching DNA patterns to be confirmed by computer assisted image analysis over the entire range of the sizing ladder. Approximately 5000 intragel and 5000 intergel comparisons were made between the restriction fragment length polymorphism (RFLP) DNA band sizes obtained from casework, mock cases, and environmentally insulted samples and the band sizes obtained from their corresponding bloodstain standards (controls). Analyses of these data suggested that fragments located in different molecular weight size regions of an analytical gel required different match criteria for assessing a visual match. The results of these analyses support the use of the following match criteria: Intragel 0.5–10 kb = ±1.7%, 10–15 kb = ±3.2%, 15–22.6 kb = ±5.8%; Intergel and blind control 0.5–10kb = ±3.0%, 10–15 kb = ±4.2%, 15–22.6 kb = ±10.0%; and human cell-line K562 and the monomorphic locus D7Z2 = ±2.5%. Each match criterion was also evaluated with respect to the distance in millimeters between matching bands throughout the 0.5–22.6 kb molecular weight size range. Applying these match criteria to different gel regions has been shown to be valid and reliable in comparisons conducted on more than 10,000 validation samples, in over 500 forensic cases and in more than 200 searches of a criminal sexual offender (CSO) database containing over 5000 individuals.
This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.
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