Environmental exposure samples. Nine bloodstains were obtained from C.T. Comey (Forensic Science Research and Training Center, F.B.I. Academy, Quantico, VA) that had been exposed to a variety of substances to which evidential bloodstains from crime scenes might have been exposed. These stains, all from the same individual (DQα genotype 1.1,4) and the substances to which each was exposed are listed in Table 1.
Forensic-type samples given to “β-test” sites. Thirty-three samples that had also been given to five different crime labs which had participated in a “β-test’ of the AmptiType HLA DQα kit were also tested using the Chelex extraction method (14). The samples and their DQα genotypes that had been obtained after organic extraction of their DNA are listed in Table 2.
Samples used in a proficiency test. Remainders or duplicates of 28 samples that had been given out as part of a blind proficiency test sponsored by the California Association of Crime Lab Directors (CACLD) were extracted using the Chelex-based procedures below and their DQα genotypes determined. The samples and their DQα genotypes, as obtained after organic DNA extraction, are listed in Table 3.
Additional bloodstains. Additional bloodstains spotted on cotton cloth were obtained from J. Maholovich (Forensic Science Associates, Richmond, CA). The DQα genotypes of these samples, determined after organic extraction of their DNA, are listed in Table 4.
le> Typing from single, stained threads
In order to test the limits of usefulness of the Chelex extraction method, two bloodstains and one semen stain previously typed were again tested. New 3 x 3-mm samples were cut from the stains. These pieces of stained fabric were then dissected into individual 3-mm yarns representing various portions of the original stains. Single, 3- mm stained yarns representing between 5.5% and 9% of the original 3 x 3-mm stains were extracted with Chelex and amplified and typed.Chelex-based extraction procedures
Note on handling Chelex solutions: When pipetting Chelex solutions, the resin beads must be distributed evenly in solution; this can be achieved by gentle mixing with a stir bar in a beaker. Make 20% and 5% (wt/vol) stock solutions in sterile, distilled water to a final volume of 100-300 ml. Add Chelex to individual samples as follows: For each set of samples, pour ca. 15 ml of Chelex stock solution into a 50-ml beaker containing a stir bar. Pipet the volume needed for each sample directly from the beaker while the stir bar is mixing. The pipette tip used must have a relative ly large bore- 1000-µl pipettor tips are adequate.HLA DQα amplification and typing
The HLA DQα sequence in samples were amplified using the AmpliType HLA·DQα kit (Cetus; 1) following the manufacturers directions. The cycling reaction was done in a programmable heat block (DNA Thermal Cycler, Perkin·Elmer Cetus, Norwalk. CT) set to heat at 94°C for 60 s (denature), incubate at 60°C for 30 s (anneal), and incubate at 72°C for 30 s (extend) by the “step-cycle’’ program. After 32 cycles, the samples were incubated an additional 10 min at 72°C.Determination of the DQα alleles present in samples was done using the immobilized, allele-specific oligonucleotideprobe system (10) provided in the kit.