A new pentaplex PCR system for forensic casework analysis.

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.