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TWGDAM Validation of AmpFISTR ProfilerÔ

The AmpFISTR ProfilerÔ PCR Amplification Kit simultaneously amplifies the following nine short tandem repeat loci: D3S13S8, vWA, FGA, TH0I, TPOX, CSFlPO, D5S818, D13S317, and D7S820. Additionally, gender determination is provided through amplification of a segment of the X-Y homologous gene amelogenin. One primer of each locus-specific primer pair is labeled with either the 5-FAM, JOE, or NED NHS-ester dye, detected as blue, green, and yellow, respectively, on ABI PRISMÔ Instruments. The probability that alleles of two unrelated individuals match by chance (Pm) at all nine STRs for African Americans (n=l95) and U.S. Caucasians (n=200) is 1 in 8 billion and 1 in 3.6 billion, respectively. Through a procedural validation process outlined by the Technical Working Group on DNA Analysis Methods (TWGDAM), critical aspects of DNA typing using the AmpFISTR ProfilerÔ kit were identified.

In collaboration with the Santa Clara County District Attorney Crime Laboratory's DNA Unit, San Jose, CA, the ability of AmpFISTR ProfilerÔ to reliably provide genotypic information under diverse conditions was assessed. Samples of dried and liquid body fluids (Reproducibility) and various biological samples from multiple individuals (Standard Specimens) were processed. DNA isolated from each of the different tissues/fluids from each individual yielded the same results. Samples were also generated to resemble forensic specimens exposed to adverse environmental conditions. These include enzymatically degraded DNA, blood and semen stains of various ages, blood and semen stains exposed to different temperatures or to sunlight (Environmental Studies). Reduction in PCR product yield reflected as the level of DNA degradation, as observed by agarose gel electrophoresis. The locus first influenced by DNA degradation was CSFlPO, the locus with the longest allele sizes, followed by D7S820, then FGA, and so forth, in order of decreasing allele length.

Presence of increasing amounts of hematin, a PCR inhibitor, in AmpFISTR ProfilerÔ amplifications caused some locus dropout, in a characteristic pattern. Influence(s) of fourteen substrate materials on amplification of blood and semen stains (1 week and 1 year timepoints) were examined (Matrix Study). Possible benefit(s) of amplifying, each locus separately (single-plexing), as opposed to multiplexing, were not observed, even with degraded DNA or in the presence of hematin.

Proficiency of the AmpFlSTR ProfilerÔ system in detection of component parts of samples containing DNA from more than one individual (Mixed Specimen Study) was investigated through typing of purified DNA mixtures and of mixtures of different body fluids (blood/blood, semen/blood, semen/saliva, saliva/blood). Adjudicated sexual assault case samples were successfully typed (Nonprobative Evidence). Characterization of stutter bands and peak height ratios were performed to assist in recognition and interpretation of samples contributed by more than one donor.

DNA types of the AmpFISTR ProfilerÔ STR loci were only obtained from primate source stains. Effects of adding amounts of input DNA outside the recommended range (1.0 to 2.5 ng) were investigated (Minimum Sample). Excess input DNA can result in increased PCR product yield, causing off-scale data and/or incomplete 3' A nucleotide addition, while extremely low input DNA may exhibit stochastic fluctuation in the ratio of alleles of a heterozygote. Definition of the limitations of the AmpFlSTR ProfilerÔ system, using optimized reagents and protocols, has culminated in the determination that DNA typing results are obtained with the reliability, sensitivity, and specificity that are mandatory in forensic analysis.

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