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Simultaneous amplification of Short Tandem Repeats (STR) loci is increasingly employed in forensics to generate discriminating genetic profiles. We have optimized rapid, single-tube DNA preparation protocols for a variety of frequently-encountered forensic-like samples. The aim was to obtain material amenable to multiplex amplification and fluorescence-based detection.

DNA was prepared from bloodstains and sperm stains on cotton, buccal swabs, hair (combed or pulled) as well as cigarette butts, envelope flaps and small stains on clothes. Samples were incubated in NP40/Tween buffer plus various additives (proteinase K, DTT, NaOH) or subjected to an organic extraction protocol. Differential DNA preparation procedures were also devised for vaginal washes containing traces of male fraction or mixed stains (blood-sperm). Yield was quantified by slot blot and hybridization with probe D17Z1. Aliquots were subjected to multiplex amplification and detection on ABD 377 Genescanners.

For all sample types, the optimal, ‘rapid protocol’ preparation could be carried out in one to three hours and found to provide a yield comparable or superior to that obtained by organic extraction. Bloodstains and swab samples required proteinase K. Incubation with DTT (low concentration) prior to boiling with NaOH was optimal for sperm-containing samples. Successful amplification and profile analysis were achieved with optimum preparations with sensitivity limits similar to organic preparations. Two sequential proteinase K digestions of the female fraction improved the resolution of male and female profiles in differential DNA extractions. Modification of amplification conditions could also improve signal quality.

High quality profiles can be obtained from rapidly prepared DNA originating from a wide variety of samples. A composite differential protocol is effective to resolve profiles from sperm-containing mixtures. The single-tube approach is economical and reduces sample loss when the amount of material is limited. Furthermore, the risk of sample mix-up is decreased.