Once treated, PCR amplification mix was added to the well and amplification performed. The species cross-reactivity study was performed using a number of commercially available non-human DNAs. Ten nanograms of each domestic animal or microbial species was amplified in duplicate. Species samples included chicken, pig, mouse, bovine, cat, dog, rabbit, deer, horse, Escherichia coli, Enterococcus faecalis, Lactobacillus acidophilis, Streptococcus mutans, Staphylococcus epidermidis,

Micrococcus luteus, Fusobacterium nucleatum, Streptococcus salivarius, Streptococcus mitis, Acinetobacter lwoffi, Pseudomonas aeruginosa, Candida albicans, and Saccharomyces cerevisiae. Three primate species, chimpanzee (male; Coriell Institute), macaque (male; Coriell GSK-3 activation Institute), and gorilla (gender unknown; privately obtained), were evaluated using 500 pg. The sensitivity Sirolimus supplier study utilized two DNA dilution series provided to all test sites. Test quantities included 500 pg, 200 pg, 100 pg, and 50 pg. An inhibitor study evaluated hematin (Sigma–Aldrich), humic acid (Sigma–Aldrich), tannic acid (Sigma–Aldrich), and EDTA (Sigma–Aldrich) titrations. Each inhibitor study site prepared its own extracted DNA, inhibitor stocks and dilutions. Two mixture series, one male–male and one male–female, were prepared and distributed. Mixture ratios included 0:1, 1:19, 1:9, 1:5, 1:2, 1:1, 2:1, 5:1, 9:1, 19:1, and 1:0 for each series. The

total template quantity was 500 pg per reaction. Concordance was performed with extracted DNA from 652 unrelated individuals from Caucasian, Hispanic, African-American, and Asian-American ethnic groups. Reaction volume studies used 1.2 mm punches of blood on Indicating FTA® cards, in addition to buccal Indicating FTA®

cards described previously. Amplification reactions were performed at 25 μl volumes on a GeneAmp® PCR System 9700 thermal cycler using a 96-well silver or gold block and max ramp rates as described in the PowerPlex® Fusion System Technical Manual [9], unless otherwise noted. The thermal cycling method for extracted DNA samples was: 96 °C for 1 min; 30 cycles of 94 °C for 10 s, 59 °C for 1 min, Tacrolimus (FK506) and 72 °C for 30 s, followed by a 60 °C final extension for 10 min and a 4 °C soak. The cycle number and final extension hold time was modified for solid support materials due to the substantial increase in template amount with these materials. FTA® card punches were amplified for 27 cycles, swab lysates were amplified for 27 or 25 cycles, and treated nonFTA punches were amplified for 25 or 26 cycles. All amplification reactions with solid support substrates utilized a 20 min final extension. Further cycle number optimization was evaluated in a cycle number study. Within that study extracted DNA samples were amplified for 29, 30, and 31 cycles, FTA® card punches for 26, 27, and 28 cycles, and treated nonFTA punches for 25, 26, and 27 cycles.