A new pentaplex PCR system for forensic casework analysis

Abstract

In 1998 the Federal Criminal Police Office of Germany (BKA) established a central genetic database of offenders and suspects to facilitate comparisons with biological samples from future criminal offences. The five obligatory short tandem repeat (STR) loci in this database (TH01, SE33, vWA, FGA and D21S11) were co-amplified in a new PCR pentaplex analysing system together with the sex-specific locus amelogenin. Due to overlapping fragment sizes, amplification products were fluorescent dye-labelled with different colours, separated by electrophoresis and detected directly using the ABI PRISM 310 Genetic Analyzer. Reproducible and reliable results were obtained from as low as 125 pg template DNA, indicating high specificity and sensitivity of the assay. Environmental studies and enzymatic digest with DNase I revealed an excellent stability of the pentaplex system with typeable results even in cases of partially degraded DNA. Complete and reproducible DNA typing was possible in blood-stain mixtures with the minor component as low as 10%. Mean stutter peak intensities were analysed for all loci and ranged from 2.7 +/- 0.8% (TH01) to 10.6 +/- 1.6% (vWA) of the main signal intensity. Allele frequencies were determined in a North Bavarian population sample (n = 121). The combination of five systems resulted in a mean exclusion chance of 99.86% and a power of discrimination of 99.999996%. No deviation from Hardy-Weinberg equilibrium could be found.

Development of a 13-locus PCR multiplex system for paternity testing

“…Therefore, the overall sensitivity of the developed system turned out to be slightly higher than that of the PowerPlex16 system (>250 pg [22]) and the AmpFlSTR Profiler Plus/AmpFlSTR Cofiler multiplex systems (>160 pg [23]). A similar sensitivity (about 125 pg) was reported for some smaller multiplex systems like the AmpFISTR Blue PCR amplification kit with three loci [9] or a pentaplex PCR system [24]. Incomplete profiles were observed using less than 100 pg template DNA, however, typing of these samples was complicated by stochastic effects, resulting in a pronounced heterozygote allelic imbalance and allelic drop-out phenomena.…”

Validation of the multiplex kit genRES MPX-2 for forensic casework analysis

“…Therefore, the overall sensitivity of the genRESMPX-2 kit was at least as high as that of the PowerPlex 16 system (>250 pg, Finis 2001), the AmpFlSTR Profiler Plus/AmpFlSTR Cofiler multiplex system (>160 pg, LaFountain et al 2001), the AmpFlSTR Blue amplification kit (>250 pg, Wallin et al 1998) and the MPX-1 multiplex kit (>125 pg, Lederer et al 2000). Nevertheless, preferential amplification and low signal intensities of 100-150 rfu were partly observed applying DNA levels below 200 pg.…”

“…In the past a variety of different PCR multiplex systems were established for forensic purposes, which allow the simultaneous amplification of different STR (short tandem repeat) loci and which are a powerful and rapid method for individual identification (Cotton et al 2000;Lederer et al 2000;Frank et al 2001). Such systems allow the reduction of time, cost and material and, therefore, permit the maximization of the forensic expressiveness even in cases of limited evidential material (Kimpton et al 1993;Urquhart et al 1995).…”

INFLUENCE OF TEMPLATE DNA DEGRADATION ON THE GENOTYPING OF SNPs AND STR POLYMORPHISMS FROM FORENSIC MATERIALS BY PCR

“…Imbalance of STR allele in the amplification using degraded DNA is also reported by others 5,26) . Allele drop in the amplification of STRs from highly degraded DNA has been reported by many researchers 2,5,18,22,23,25) . It can be speculated that, when allelic imbalance was observed, the size of amplifiable template DNA might be close to the PCR product size.…”