The art of reading sequence electropherograms.
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Artifacts↗
Biomedical subjects
W Parson
Publications and source records attributed to W Parson.
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.
Adolescent↗
Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.
Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.
Analysis of Variance↗
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.
Amelogenin↗
More evidence for non-maternal inheritance of mitochondrial DNA?
BACKGROUND: A single case of paternal co-transmission of mitochondrial DNA (mtDNA) in humans has been reported so far. OBJECTIVE: To find potential instances of non-maternal inheritance of mtDNA. METHODS: Published medical case studies (of single patients) were searched for irregular mtDNA patterns by comparing the given haplotype information for different clones or tissues with the worldwide mtDNA database as known to date-a method that has proved robust and reliable for the detection of flawed mtDNA sequence data. RESULTS: More than 20 studies were found reporting clear cut instances with mtDNAs of different ancestries in single individuals. As examples, cases are reviewed from recent published reports which, at face value, may be taken as evidence for paternal inheritance of mtDNA or recombination. CONCLUSIONS: Multiple types (or recombinant types) of quite dissimilar mitochondrial DNA from different parts of the known mtDNA phylogeny are often reported in single individuals. From re-analyses and corrigenda of forensic mtDNA data, it is apparent that the phenomenon of mixed or mosaic mtDNA can be ascribed solely to contamination and sample mix up.
DNA, Mitochondrial↗
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.
Alleles↗
Phytosterols in nonfat and low-fat beverages have no impact on the LDL size phenotype.
OBJECTIVE: To examine the impact of nonfat and low-fat phytosterol-enriched beverages on low-density lipoprotein (LDL) electrophoretic characteristics. DESIGN: Double-blind, randomized, crossover, placebo-controlled dietary trial. SETTING: Diets were prepared and consumed at the Mary Emily Clinical Nutrition Research Unit of McGill University. Analyses were performed at the Institute on Nutraceuticals and Functional Foods of Laval University. SUBJECTS AND INTERVENTION: In total, 15 moderately hypercholesterolemic persons consumed each of three experimental diets that each comprised a different beverage: nonfat placebo (NF control), nonfat with phytosterols (NFPS) or low-fat with phytosterols (LFPS). Participants consumed three beverages daily at meal time for a total of 1.8 g of phytosterols per day. Nondenaturing 2-16% polyacrylamide gradient gel electrophoreses were used to characterize LDL size characteristics. RESULTS: The NFPS and LFPS beverage induced no significant changes in several features of the LDL size phenotype compared to the control diet. CONCLUSION: The consumption of phytosterol-supplemented nonfat and low-fat beverages is not associated with clinically meaningful changes in the LDL particle size phenotype.
Adult↗
Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.
A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.
DNA, Mitochondrial↗
A proposed nomenclature for 15 canine-specific polymorphic STR loci for forensic purposes.
We performed a population study on 15 polymorphic STR loci (FH2010, FH2079, PEZ2, VWF.X, FH2054, FH2087Ub, FH2611, WILMS-TF, PEZ12, PEZ15, PEZ6, FH2087Ua, ZUBECA4, ZUBECA6, FH2132) on 131 randomly selected dogs. Alleles were identified and grouped according to their estimated fragment length using fixed allelic bins encompassing one base-pair. The allele assignment was confirmed by sequence analysis of homozygote and cloned heterozygote alleles. In order to develop a uniform repeat-based nomenclature, extensive sequence analysis was performed on a selection of alleles from each STR locus. The proposed nomenclature refers to the internationally recognised recommendations for human-specific STR loci in forensic applications. The 15 canine-specific STR loci were grouped into 3 classes (simple STRs, compound STRs and complex/hypervariable STRs) according to their complexity and variability within the repeat structure. Finally, we evaluated the precision of fragment size estimation on a capillary electrophoresis platform and demonstrated reproducibility of fragment length estimation for single base-pair intermediate alleles.
Alleles↗
New sensitive amplification primers for the STR locus D2S1338 for degraded casework DNA.
PCR primers for the locus D2S1338 were redesigned in order to reduce the length of the amplification product compared with the conventional design. The new amplification primers were applied to highly degraded casework samples, which gave no or only weak results for D2 in previous analyses. The application of the new primers resulted in an increased overall typing success rate. In a concordance study between the conventional and the newly designed primers, no genotype differences were revealed in 503 randomly selected individuals. This suggests robust amplification conditions with respect to mutation-based allele drop-out.
DNA Fingerprinting↗
Population genetic comparisons of three X-chromosomal STRs.
The X-chromosomal short tandem repeats (STRs) DXS6800, DXS101 and DXS8377 were analysed in male and female population samples from Germany and Austria using a PCR multiplex approach. We investigated 135 family trios from Innsbruck (Austria) and surrounding areas and 50 families and further male and female samples from Ulm (Germany) and surrounding areas. The comparisons of the allele frequencies gave similar distributions for Innsbruck and Ulm although minor variations were found for some alleles. Additionally, some differences were found when comparing the allele frequencies of the male and female samples independently. The forensic efficiency values demonstrate that especially DXS101 and DXS8377 are highly informative markers for kinship analysis and deficiency cases. Based on the investigated meiotic events no new mutations were detected.
Adolescent↗
Loss of zidovudine related mutations in the reverse transscriptase gene of HIV after switching therapy.
The standard treatment for HIV infected patients is the highly active antiretroviral therapy. Due to resistance developments treatment failure can be found in some patients. In our study two different strategies are compared, which may reduce resistance mutations. Six patients (group A and B) have been monitored for about six years. Group A patients had a switch in their AZT-containing treatment to non AZT-containing regimens. In group B patients AZT-containing regimens' were interrupted by drug holidays. Early mono- and dual-therapies containing zidovudine (AZT) have been applied in all patients with poor long-term improvements. Due to the rapid development of escape mutants viral rebound was observed soon after treatment initiation. Genotypic assays were developed to asses AZT-resistance mutations. The longer AZT had been applied the more mutations had developed. These mutations disappeared when patients were taking "drug-holidays" and drug selection pressure was missing. Besides, it was demonstrated in two patients that these AZT-mutations could disappear, if in combination therapies AZT was replaced by other antiretroviral drugs. This study shows that not only by drug-holidays but also by switches in therapy mutations can disappear, which is especially important for patients with low CD 4 cell counts and high viral load levels.
Anti-HIV Agents↗
Rare failures in the amelogenin sex test.
Determination of sex using the amelogenin sex test is well established in the forensic field especially for casework and DNA databasing purposes. The sex test is part of commercially available PCR kits. Among 29,432 phenotypic male individuals stored in the Austrian National DNA database, 6 individuals were found to lack the amelogenin Y-specific PCR product which was confirmed using alternative amelogenin primers. The amplification of eight Y-chromosomal STR markers resulted in full profiles in five out of the six samples, one sample failed to amplify Y-STRs at all. The amplification of a fragment of the SRY gene gave positive results in all six samples, confirming the male phenotype of the individuals. The observed failure rate of the amelogenin sex test was 0.018% in this study.
Amelogenin↗
STR loci Penta D and Penta E: Austrian Caucasian population data.
Allele frequencies for the short tandem repeat (STR) loci Penta D and Penta E, included in the PowerPlex 16 kit were obtained from a sample of 269 unrelated Austrian Caucasian individuals. Both loci met Hardy-Weinberg expectations.
Austria↗
Gabon black population data on the ten short tandem repeat loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA.
Allele frequencies for ten short tandem repeat (STR) loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01 and FGA were determined in a Black African sample population from Gabon. All loci were highly polymorphic and except for TH01, D21S11 and D16S539, all met Hardy-Weinberg expectations. There was little evidence of association of alleles between the loci in this database. The combined power of exclusion for the ten STR loci was 0.999981. While significant differences between the Gabon population and the Austrian Caucasian population were found at all loci, significant differences were found between the Gabon population and Zimbabweans only for D3S1358 and between the Gabon population and African Americans only for TH01 and D8S1179.
Black People↗
Identification of members of the Aspergillus nidulans SREA regulon: genes involved in siderophore biosynthesis and utilization.
Iron is an essential trace element for almost all organisms. However, an excess of this metal within cells can be deleterious on account of catalysis of cell-damaging hydroxyl radicals. Therefore, the concentration of iron within cells is tightly regulated and the primary control occurs by regulating its uptake. Under conditions of low iron availability, most fungi mobilize extracellular iron by excretion of low-molecular-mass ferric iron chelators, termed siderophores. Due to the potential impact of iron metabolism on fungal pathogenicity, a better insight into siderophore-mediated iron uptake is needed. In Aspergillus nidulans, siderophore biosynthesis and uptake are negatively regulated by the GATA-type transcription factor SREA. Hence, genes involved in siderophore biosynthesis and uptake are characterized by transcriptional induction under iron limitation in wild-type strain and de-repression in an sreA-deletion strain under conditions of sufficient iron supply. Such genes have been searched for using different strategies, e.g. differential mRNA display and expression analysis of candidate genes from various A. nidulans sequence databases. The identified genes presumably encode enzymes needed for siderophore biosynthesis, and transporters involved in siderophore uptake and/or excretion. The functional characterization of these genes will help to unravel the pathways involved in siderophore biosynthesis and uptake.
Aspergillus nidulans↗
Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles.
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DNA Fingerprinting↗
Analysis of polymerase chain reaction products by on-line liquid chromatography-mass spectrometry for genotyping of polymorphic short tandem repeat loci.
Capillary ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was used to separate and purify DNA fragments amplified by the polymerase chain reaction (PCR) prior to their characterization by electrospray ionization mass spectrometry (ESI-MS). The investigation by ESI-MS of single- or double stranded species could be effortlessly selected by chromatography of the nucleic acids under either nondenaturing or denaturing conditions, which were realized by proper adjustment of the column temperature. ESI-MS detection sensitivity was improved by a factor of 10 upon replacement of 25 mM triethylammonium bicarbonate as ion-pair reagent by 25 mM butyldimethylammonium bicarbonate because of the applicability of higher acetonitrile concentrations to elute the DNA from the monolithic, poly(styrene/divinylbenzene)-based capillary columns. For fragments ranging in size from 67 to 84 base pairs, the mass accuracies and mass reproducibilities were typically better than 0.02 and 0.008%, respectively, which enabled the characterization and identification of the PCR products with high confidence. The hyphenated method was applied to the genotyping of polymorphic short tandem repeat (STR) loci from the human tyrosine hydroxylase gene (humTH01). The different alleles both in homo- and heterozygotes were identified on the basis of the masses of the single-stranded amplicons and were in full accordance with the alleles identified by conventional capillary electrophoretic sizing.
Alleles↗