Petra Grubwieser

A new "miniSTR-multiplex" displaying reduced amplicon lengths for the analysis of degraded DNA

A multiplex PCR was designed for the loci D2S1338, D16S539, D18S51, TH01 and FGA using redesigned primers in order to reduce the lengths of the amplification products compared to the designs used in commercially available multiplex PCR kits, also including amelogenin. The new PCR primers were used to amplify highly degraded DNA from casework samples, which had shown no or only poor results for these loci in previous analyses with standard primer sets. The application of the new miniSTR-multiplex resulted in an increased overall typing success rate for degraded DNA samples. In a concordance study between the conventional and the newly designed primers, no genotype differences were revealed in 124 randomly selected individuals.

A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA.

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs)--generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets--enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

Y-STR typing of an Austrian population sample using a 17-loci multiplex PCR assay

Y-chromosomal STR haplotypes were determined from a sample of 135 unrelated men and 70 sons from Tirol (Austria) using the AmpFlSTR Yfiler PCR amplification kit (Applied Biosystems) that coamplifies 17 Y-STRs. The panel of markers includes the 9-loci European minimal haplotype (minHt) and, in addition, the markers DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA C4) and Y GATA H4. A total of 130 different haplotypes (125 were unique) were identified by the 17 Y-STR markers, an increase of 19 compared with the minHt. The gene diversity of DYS635, DYS456 and DYS458 exceeded 0.75 and only that of the duplicated marker DYS385 (0.86) was higher. Consistently high haplotype diversity values were found in all tested Y-SNP haplogroups. Because the simultaneous analysis of 17 Y-STR systems offers a high power of discrimination at minimum sample consumption, the Yfiler kit is a promising tool for forensic applications.

Evaluation of an extended set of 15 candidate STR loci for paternity and kinship analysis in an Austrian population sample

We investigated 15 polymorphic short tandem repeat (STR) loci (D1S1656, D7S1517, D8S306, D8S639, D9S304, D10S2325, D11S488, D12S391, D14S608, D16S3253, D17S976, D18S1270, D19S253, D20S161, and D21S1437) which are not included in the standard sets of forensic loci. The markers were selected according to the complexity of the polymorphic region: Of the 15 investigated loci, 7 loci showed a simple repeat structure (D9S304, D10S2325, D14S608, D16S3253, D18S1270, D19S253, and D21S1437), 3 loci (D7S1517, D12S391, and D20S161) consisted of compound repeat units, and 5 loci (D1S1656, D8S306, D8S639, D11S488, and D17S976) showed a more complex polymorphic region partly including different repeat blocks and incomplete repeat units, which resulted in a relatively high proportion of intermediate alleles. A population study on a sample of 270 unrelated persons from Austria was carried out. We did not observe significant deviations from Hardy–Weinberg expectations. The combined probability of exclusion for the 15 loci was 0.99999998. In combination with the conventional set of STR markers included in commercially available kits (no linkage was observed between these 15 loci and the Powerplex 16 System loci), these markers are approved as highly discriminating forensic tools, also suitable for the analysis of difficult paternity and kinship constellations.

Unusual variant alleles in commonly used short tandem repeat loci

Unusually large variant alleles were observed in the short tandem repeat (STR) systems D3S1358 and D21S11, both of which are included in the international standard set of loci (ISSOL) and routinely typed in National DNA intelligence databases worldwide. The observed alleles fell within the size range of the adjacent STR marker, which could easily cause problems with respect to correct allele assignments for both loci concerned. We compared the amplification and potential interpretation with three different commercially available kits, which are frequently used in forensic work. PCR products were cloned and sequenced in order to determine the structure of these unusual allele variants and confirm their size and designation (D3S1358 allele 26, D21S11 allele 46). In the locus D21S11 we observed an as yet undescribed partial duplication of the constant region.

Characterization of mtDNA SNP typing and mixture ratio assessment with simultaneous real-time PCR quantification of both allelic states

We performed a study on the forensic utility of allele-discriminatory quantitative real-time PCR (rtPCR) using Minor Groove Binder TaqMan probes, targeting the highly variable mitochondrial single nucleotide polymorphism 16519T/C. The apparent single-cycle PCR efficiency was virtually 100% for both 16519 alleles. The allele designations made by rtPCR were concordant with the results obtained in a previous study by sequencing analysis. In heteroplasmic samples, minor allele proportions down to 9% were unambiguously detected and quantified. The variation in allele proportion estimates was essentially the same within and between different rtPCR runs, and the differences between total copy number estimates found for rerun samples were comparable to those found with non-allele-discriminatory quantitative rtPCR assays.

BAK-AAK-Quotient im Konzentrationsbereich von 0,5‰ (0,25 mg/l Ausatemluft) Eine Trinkversuchserie mit dem geeichten Atemalkoholtestgerät Alcotest 7110 MK III A (Österreich-Ausführung)

ZusammenfassungZiel der vorliegenden Arbeit war es mit dem in Österreich sei 1997 zugelassenen Atemalkoholmessgerät Alcotest 7110 MK III A von Dräger, das sich technisch in einigen Punkten von dem in Deutschland zugelassenen Alcotest 7110 MK III Evidential unterscheidet, unter realitätsnahen Bedingungen Daten für eine Konversionsfaktor Atemalkoholkonzentration-Blutalkoholkonzentration (AAK-BAK) zu ermitteln und das Messverhalten dieses für Österreich spezifischen Geräts in dem Konzentrationsbereich um 0,25 mg/l Ausatemluft zu evaluieren. In 32 Trinkversuchen wurden bei 139 Probanden 30 min nach Trinkende zeitgleich AAK und BAK bestimmt. Die AAK-Werte lagen zwischen 0,05 und 0,78 mg/l Ausatemluft, die BAK-Werte zwischen 0,14 und 1,72‰ (g/l). Die daraus errechneten Konversionsfaktoren lagen zwischen minimal 1.380 und maximal 2.720 bei einem Mittelwert von 2.062. Wegen dieser großen Variationsbreite muss für die Beurteilung einer Beeinträchtigung durch Genussalkohol BAK-Werten der Vorzug gegeben werden. Sollten nur AAK-Werte vorliegen, können nach unseren Daten für die Umrechnung von AAK in BAK bei einer angebenden statistischen Wahrscheinlichkeit von 95% ein minimaler Konversionsfaktor von 1.631 und ein maximaler Konversionsfaktor von 2.493 verwendet werden.AbstractIn this study the analytical performance of the breath alcohol analyser Alcotest MK III A from Dräger was evaluated in the concentration range of 0.25 mg/l to collect data for the determination of the blood alcohol-breath alcohol (BAC-BrAC) quotient. This breath testing device has been approved for use in Austria since 1997 and differs in some technical aspects from the Alcotest 7110 MK III Evidential, which is approved for use in Germany. In 32 controlled experiments the BAC and BrAC of 139 participants were determined simultaneously 30 min after the last consumption of alcohol. The measured BAC and BrAC values ranged between 0.14 and 1.72‰ (g/l), and 0.05 and 0.78 mg/l, respectively. A conversion factor (CF) between BAC and BrAC was determined from these data pairs with a minimum of 1,380 and a maximum 2,720 and a mean value of 2,062. Due to the large variability of the CF, analysis of BAC is preferable to using conversion calculations from a forensic point of view. According to our data, if only BrAC values are available a CF ranging from 1,631 (min) and 2,493 (max) can be used to convert BrAC to BAC with a statisticl significance of 95%.