ABSTRACT
The present study deals with low-volume amplification of short tandem repeats (STRs) for forensic DNA analysis. A chemically structured chip in microscopic slide format was used to amplify standard forensic casework samples in a 1-microL reaction volume primarily with the well-known and widely used PowerPlex kit and with other commercially available STR kits. Tests regarding sensitivity, mixture analysis, robustness, reproducibility, buffer composition and technical performance were carried out to check the usefulness of this amplification strategy. The results obtained show that low-volume amplification is a promising option in the forensic DNA typing toolbox. Restrictions to this method, which are strictly related to the kit and the respective buffer used, were found in low copy number (LCN) DNA typing, mixture analysis and technical performance. Problematic typing results included artefact alleles, increase in locus and heterozygote imbalance, allelic and locus dropout as well as increase of stutters, especially when amplifying less than 200-300 pg of DNA. In contrast, convincing advantages are a higher sensitivity, better amplification efficiency and the low cost factor of this method.
Subject(s)
Microsatellite Repeats/genetics , Nucleic Acid Amplification Techniques , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Blood Stains , Humans , Mouth Mucosa/metabolism , Predictive Value of TestsABSTRACT
In forensic DNA analysis, improvement of DNA typing technologies has always been an issue. It has been shown that DNA amplification in low volumes is a suitable way to enhance the sensitivity and efficiency of amplification. In this study, DNA amplification was performed on a flat, chemically structured glass slide in 1-microl reaction volumes from cell line DNA contents between 1,000 and 4 pg. On-chip DNA amplification reproducibly yielded full allelic profiles from as little as 32 pg of template DNA. Applicability on the simultaneous amplification of 15 short tandem repeats and of a segment of the Amelogenin gene, which are routinely used in forensic DNA analysis, is shown. The results are compared to conventional in-tube amplification carried out in 25-microl reaction volumes.
