DNA EXTRACTION STRATEGIES FOR AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS

A polymerase chain reaction-based DNA typing method, amplified fragmen t length polymorphism (AMP-FLP) analysis, has shown promise as a means of analyzing forensic biological evidence. A variety of DNA extractio n methods were evaluated for their suitability for AMP-FLP analysis. F actors that were considered in the evaluation included DNA yield, abil ity of DNA to be amplified, the presence of DNA fragments other than t hose expected for the alleles in the sample, and differential amplific ation of different sized alleles for a sample. An initial screen of ei ght extraction methods was conducted on bloodstains deposited on cotto n sheeting. These methods included Chelex(R) 100, organic extraction f ollowed by Centricon 100(R) (Amicon, Inc., Beverly, MA) dialysis and c oncentration, Geneclean(TM) (Bio 101, La Jolla, CA), GlassMax(TM) colu mns (Gibco BRL, Gaithersburg, MD); GlasPac(TM) (National Scientific Su pply Co., Inc., San Rafael, CA), Qiaex (Qiagen Inc., Chatsworth, CA), Elu-Quik(TM) (Schleicher and Schuell, Keene, NH), and DNA Capture Reag ent (Gibco BRL, Gaithersburg, MD). Then, four methods, Chelex(R) 100 e xtraction, organic extraction followed by ethanol precipitation, organ ic extraction followed by Centricon 100(R) (Amicon, Inc., Beverly, MA) dialysis and concentration, and Geneclean were evaluated on blood and semen stains. These stains were deposited on a variety of substrates, including cotton sheeting, denim, wallboard, nylon, wood, and carpet. The effect of addition of bovine serum albumin (BSA) to the amplifica tion reaction was also examined. The method judged most suitable for A MP-FLP analysis was organic extraction followed by Centricon 100(R) di alysis and concentration, with BSA added to the amplification reaction . Additionally, a modification of an existing differential extraction procedure for separating non-sperm from sperm DNA was developed.