A single direct amplification method for forensic casework references on a variety of substrates

Abstract

A single direct amplification method for buccal and blood reference samples, on different substrates, containing a wide range of cellular material, was developed. This method uses the GlobalFiler™ STR kit, which was designed for amplification of previously purified and quantified DNA. Buccal samples on cotton swabs, Bode collectors, and Whatman® FTA® cards, and liquid blood, blood on Whatman® 903 and FTA® cards, and autopsy bloods were examined. A first pass success rate, that is, complete concordant profiles with no GeneMapper™ ID-X quality flags, of > 95% was obtained. This method involves the lysis of reference samples (tips of cotton swabs, double 4.8 mm diameter Bode or FTA® buccal punches, and single 4.8 mm diameter blood punches on Whatman® 903 or FTA® cards) in 100 µl of Prep-n-Go™ lysis buffer, and amplification of 2 µl of the lysate with 25 cycles. Data collection is optimized by use of the spectral pull-up reduction and off-scale recovery features of the AB3500 Genetic Analyzer Data Collection version 4 software, and by injection of PCR products for two different lengths of time (15 and 7 s). Data was analyzed with GeneMapper™ ID-X version 1.6, which allowed an increased signal dynamic range (maximum peak height of 64000 RFU), and by employing a 10% global filter, and a 20% global stutter filter. Complete GlobalFiler™ profiles were generated from as little as 5 µl of blood, 1 buccal swipe on cotton swab and Bode collectors, and collection of buccal cells deposited on the foam heads of EasiCollect™ FTA® collectors for 5 s.