DNA Extraction Strategies for Amplified Fragment Length Polymorphism Analysis

Abstract

A polymerase chain reaction-based DNA typing method, amplified fragment length polymorphism (AMP-FLP) analysis, has shown promise as a means of analyzing forensic biological evidence. A variety of DNA extraction methods were evaluated for their suitability for AMP-FLP analysis. Factors that were considered in the evaluation included DNA yield, ability of DNA to be amplified, the presence of DNA fragments other than those expected for the alleles in the sample, and differential amplification of different sized alleles for a sample. An initial screen of eight extraction methods was conducted on bloodstains deposited on cotton sheeting. These methods included Chelex® 100, organic extraction followed by Centricon 100® (Amicon, Inc., Beverly, MA) dialysis and concentration, Geneclean™ (Bio 101, La Jolla, CA), GlassMax™ columns (Gibco BRL, Gaithersburg, MD); GlasPac™ (National Scientific Supply Co., Inc., San Rafael, CA), Qiaex (Qiagen Inc., Chatsworth, CA), Elu-Quik™ (Schleicher and Schuell, Keene, NH), and DNA Capture Reagent (Gibco BRL, Gaithersburg, MD). Then, four methods, Chelex® 100 extraction, organic extraction followed by ethanol precipitation, organic extraction followed by Centricon 100® (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 amplification reaction was also examined. The method judged most suitable for AMP-FLP analysis was organic extraction followed by Centricon 100® dialysis 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.