Twgdam Validation of Ampf_str•: PCR Amplification Kits for Forensic DNA Casework

Abstract

Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFᐉSTR™ PCR Amplification Kits (i.e., AmpFᐉSTR Green I, Profiler™, Profiler Plus™, and COfiler™ kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFᐉSTR locus amplification did not offer consistent benefit over AmpFᐉSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFᐉSTR PCR Amplification Kits reproducibly yielded sensitive and locusspecific results, as required in routine forensic analyses.

Magnitude-dependent variation in peak height balance at heterozygous STR loci

“…STR typing typically involves a polymerase chain reaction (PCR) amplification step followed by size fractionation of the resulting products and fluorescent signal detection and processing. Numerous studies have shown that the signal associated with each allele of a heterozygote at a given locus is approximately equal [3][4][5][6][7][8][9]. General practice has shown that the peak height ratio, as measured by dividing the height (in relative fluorescent units (RFUs)) of the lower quantity allele by the height of the higher quantity allele "should be greater than approximately 70% in a single source sample" [3].…”

Untitled

“…In forensic geneticists, there are regions within the genome that are hypervariable and these have been the target for most forensic analysis. 7 Commonly used regions include sequences that are repeated tandemly. The original DNA "fingerprinting" analysed mini-satellites are often referred to as variable number tandem repeats (VNTRs).…”

“…These include the following light (UV), humidity has more of an effect on the quality of the DNA, rather than the quantity, elevated temperatures and moisture will degrade DNA and will make it difficult to obtain a profile, fungal contamination and length of the postmortem interval. 6,7,9,10,17 The survival of DNA depends on several factors and environmental conditions, hence, the purpose of the present study was to investigate mainly the effect of various temperatures and humidity on DNA survival over time.…”

Multiplex_QA: An exploratory quality assessment tool for multiplexed electrophoretic assays

“…A number of genetic analyzer instrument platforms have been introduced over the years and validated through examination of collected data [7][8][9][10]. Currently, the forensic human identity community mostly uses CE systems that evaluate one to a few dozen samples at a time.…”