Thomas Lederer

Purification of STR-multiplex-amplified microsamples can enhance signal intensity in capillary electrophoresis.

Abstract In this study, the effect of sample purification on total signal intensities of samples amplified with genRES MPX-2 (nine-locus multiplex) prior to capillary electrophoretic analysis has been investigated. Sample purification with the Qiaquick PCR purification kit led to an increase of the relative fluorescent signal intensity by a factor of 3.8 ± 0.8. In contrast, the application of larger sample volumes led to a decrease of signal intensities from 20% to 80%, depending on whether the samples were purified or not. In addition, increase of injection time showed a linear increase of signal intensity between 3 s and 10 s. Increasing the number of PCR cycles from 30 to 33 also led to a significant increase of signal intensities. Nevertheless, this increase greatly depended on the fragment lengths and was sometimes accompanied by the appearance of non-specific signals. In combination, optimisation of sample preparation and increase of injection time may intensify signals up to 12-fold, thereby increasing the overall sensitivity of the assay. This may be of special interest for forensic analysis of microspecimens containing limited amounts of DNA.

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 bloodstain 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.

An accurate method for determining the helical repeat of DNA in solution reveals differences to the crystal structures of two B-DNA decamers

Many DNA sequences have been studied by X-ray crystallography with the goal of deciphering a sequence-structure code. We have determined the helical repeats of two B-type DNA decamers in solution employing an electrophoretic method based on phasing of bent segments. The decamers contain recognition sites for the dcm methyltransferase and for the restriction nuclease NarI with a mutational hotspot. Their helical repeats are 10.59(+/- 0.05) bp and 10.52(+/- 0.03) bp, respectively, whereas crystallographic analysis yielded 10.0 bp in the solid state. This difference is greater than that for the transition between B- and A-type DNA in solution. Thus, reliable information about the polymorphism of DNA in solution must be based on both X-ray and solution data. We describe a generally applicable approach to accurately determine helical repeats of small DNA duplexes in solution.

Characterization of two unusual allele variants at the STR locus ACTBP2 (SE33)

Two uncommon allelic variants have been observed at the locus ACTBP2 (SE33) and both were located far outside the ladder range of commercially available typing kits. The short variant showed a 60-bp deletion upstream of the 5′-flanking region with a typical type I repeat structure, which actually would have to be assigned as allele 16. The long allele revealed a typical type III sequence structure that has to be designated as allele 41.

Commentary on: Coble MD, Butler JM. Characterization of New miniSTR Loci to Aid Analysis of Degraded DNA. J Forensic Sci 2005;50:43–53.

Sir: In the above-mentioned article by Coble and Butler, STR loci were described that allow the generation of amplicons less than 125 bp in size and, therefore, particularly may be suitable for the forensic analysis of degraded DNA. In this regard, the development and evaluation of two multiplex assays are presented, including the construction of allelic ladders (1). Based on the published data, an STR PCR-assay for the locus D10S1248 was established in our laboratory. For the creation of an allelic ladder, the DNA of a total of 98 blood samples was extracted and amplified. After separation on polyacrylamide gels, different allele types were cloned into an M13 vector system and subsequently sequenced. The assignment of alleles was performed in consideration of the recommendations of the International Society of Forensic Genetics (2). For any further allelic typing, the DNA 9947A (Promega, Madison, WI) was used as a control. Although the genotype of this DNA is supposed to be 14/16 (3), our typing revealed the genotype 13/15. The same effect was observed for another control DNA (007, Applied Biosystems, Foster City, CA). Here, the genotype seemed to be 12/15 instead of 13/16, as given in Coble (3). For further clarification, the 9947A control DNA was directly sequenced. Using the above-mentioned nomenclature, the genotype consequently turned out to be 13/15. The observation of a possibly erroneous allele designation by Coble and Butler is supported by population data, which apparently have been surveyed with allelic ladders from these authors (1,4,5). Comparing different data on several population subgroups, a noticeable step in the frequency of occurrence of short alleles could be observed between allele 13 (published frequency: 0.02–0.12) and 14 (published frequency: 0.19–0.36). However, in concordance with sequencing results, this significant frequency shift was noticed between alleles 12 and 13 in our population sample. For many STR loci, the increase in information about sequence and substructure has repeatedly raised questions affecting nomenclature rules in the past. However, a uniform allelic assignment is required for interlaboratory data comparison and exchange, particularly in consideration of international databases. As the repeat structure of D10S1248 locus seems to be quite unambiguous on the bases of published sequence information (GenBank accession number AL391869), a revision of the allelic classification by Coble and Butler and, hence, a verification of population data published so far would be highly preferable.