Technical Note

The forensic community has been applying techniques of molecABSTRACT: Studies were performed as recommended by the ular biology to the analysis of DNA extracted from casework samTechnical Working Group on DNA Analysis Methods (TWGDAM) ples since the mid 1980’s. Initially, restriction fragment length committee to validate the AmpFISTR Blue PCR Amplification Kit polymorphism (RFLP) analysis (1–3) was implemented. While for forensic casework applications. The kit coamplifies the tetranuproviding excellent discrimination and mixture interpretation, cleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and RFLP analysis is time consuming, requires a relatively large detected directly using the ABI PRISMe 377 DNA Sequencer or amount (at least 20-50 nanograms) of high molecular weight DNA, the 310 Genetic Analyzer. CEPH family studies demonstrated Menand is unable to provide discrete alleles. Polymerase chain reaction delian inheritance of these loci and probability of identity values (PCR) based assays were subsequently developed (4,5), which from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all address these disadvantages. Currently, the most popular PCRstudies examining different body tissues and fluids, the expected based systems used for forensic applications are the AmpliType genotypes were observed. Studies to determine and test the PCR reverse dot-blot typing kits (6–11) and the AmpliFLPe D1S80 reagent components and thermal cycling parameters demonstrated fragment sizing kit (12–14). While these assays address the shortspecificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 comings of RFLP analysis, there are limitations to their use. ng. A variety of environmental studies were performed, as forensic Mainly, the discrimination potential of the reverse dot-blot typing samples are often exposed to different environmental conditions kits is greatly reduced relative to that with RFLP, making it more and substances which may degrade DNA or inhibit the amplification difficult to exculpate suspects and resolve mixtures. With regard process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then to the D1S80 kit, the discrimination potential of this locus can be D3S1358; this is the expected pattern according to locus size. In very informative yet the allele size range is rather large, resulting studies of PCR inhibition, the pattern in which the loci became in potential for preferential amplification. undetectable was different; FGA was the first locus to become undeIdeally, forensic assays should include high discrimination tectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, power, maximal mixture interpretation capability, high sensitivity, even in cases of degradation or inhibition. The occurrence of preferability to obtain results from degraded DNA, minimal risk of prefential amplification was very rare, particularly in noncompromised, erential amplification, discrete alleles, and high throughout potenunmixed samples. Artifact peaks were not observed in any instance. tial. In an attempt to better achieve these desirable features, the Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; forensic community has recently been exploring the utility of short the limit of detection was 1;10 for 1 ng total genomic DNA and tandem repeat (STR) polymorphic loci (15–20). STR loci contain 1;30 for 5 ng. DNA extracted from nonprobative case evidence reiterated nucleotide sequences 2 to 7 bases in length, are present was successfully amplified and genotyped. All such studies indicate in great abundance throughout the human genome, and exhibit a that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results. high degree of polymorphism in humans. STR loci are similar to