Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial

A qualitative and quantitative analytical method was developed and validated for the determination of 49 licit and illicit drugs in oral fluid. Small oral fluid samples, volume 1mL, were collected from volunteers using a modified Omni-Sal device and the analytes were extracted from an oral fluid/buffer mixture using a single Bond Elut Certify solid phase extraction cartridge. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) and gas chromatography-repetitive full scan mass spectrometry (GC-MS) were used in parallel to analyze the extracts for the targeted drugs. Extracts were analyzed by GC-MS in their underivatized form and as their pentafluoropropionyl derivatives. Deuterated internal standards were used for quantification of drugs of abuse by LC-MS-MS to minimize matrix effects. Methadone-d(9) and tumoxetine were used as the internal standards for quantification of non-derivatized and derivatized analytes respectively by GC-MS. Linearity was demonstrated over the range 5-200 ng/mL and limits of detection were less than 4 ng/mL for each drug analyzed. The method demonstrated acceptable recoveries for most of the analytes and good intra- and inter-day precision. Acquisition of data by repetitive full scan GC-MS allows the addition of further analytes to the target menu.     

Naturschutzrechtliche Vorgaben zur Verwendung gebietseigener Gehölze

Zusammenfassung  Die genetische Vielfalt ist oft übersehenes Schutzgut des Naturschutzrechts. Sie ist der Grund, warum zunehmend gefordert wird, Anpflanzungen nur mit gebietseigenem Pflanzmaterial durchzuführen. Rechtliche Probleme stellen sich hier an der Schnittstelle von Naturschutz- und Vergaberecht. Soll n?mlich bei der ?ffentlichen Auftragsvergabe zur Anpflanzung von Geh?lzen oder beim Einkauf von Pflanzmaterial nicht gegen vergaberechtliche Vorschriften versto?en werden, bedarf es hinreichender, im Naturschutzrecht verwurzelter Gründe, um ?ffentliche Ausschreibungen ausschlie?lich auf gebietseigenes Pflanzmaterial einzuengen. Diesen naturschutzrechtlichen Verpflichtungen geht der vorliegende Beitrag nach.     

Updated Brazilian genetic data, together with mutation rates, on 19 STR loci, including D10S1237

Allelic frequencies for 19 STR loci (F13B, TPOX, D3S1358, FGA, CSF1PO, D5S818, F13A01, D7S820, D8S1179, D10S1237, TH01, VWA, D13S317, FESFPS, Penta E, D16S539, D18S51, D19S253, and D21S11) were obtained from an average of 13,000 unrelated Brazilian adults undergoing parentage testing. D10S1237 is a tetranucleotide repeat locus shown to be useful for forensic and paternity studies. Null allele frequencies and mutation rates were ascertained from this population sample.     

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439)

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.     

Comparison of STR loci profiles obtained from extracted FTA discs using different DNA polymerases

Six commercially available DNA polymerases together with their respective buffers were compared as to their ability to generate profiles of commonly used short tandem repeat loci. Extracted FTA paper discs were used in two fluorescent multiplex PCRs containing the same number of units of DNA polymerase to amplify 21 STR loci and the resultant alleles were visualized after electrophoresis on a capillary sequencer. Significant differences were observed upon comparing the profiles: one polymerase failed to amplify larger alleles and presence of additional peaks varied according to the enzyme used. The use of hot-start polymerases did not affect the quality of the resultant profiles in this comparison.     

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.     

The 2000-2001 GEP-ISFG Collaborative Exercise on mtDNA: assessing the cause of unsuccessful mtDNA PCR amplification of hair shaft samples

We report the results of Spanish and Portuguese working group (GEP) of International Society of Forensic Genetics (ISFG) Collaborative Exercise 2001-2002 on mitochondrial DNA (mtDNA) analysis. 64 laboratories from Spain, Portugal and several Latin-American countries participated in this quality control exercise. Five samples were sent to the participating laboratories, four blood stains (M1-M4) and a sample (M5) consisting of two hair shaft fragments. M4 was non-human (Felis catus) in origin; therefore, the capacity of the labs to identify the biological source of this sample was an integral part of the exercise. Some labs detected the non-human origin of M4 by carrying out immuno-diffussion techniques using antihuman serum, whereas others identified the specific animal origin by testing the sample against a set of animal antibodies or by means of the analysis of mtDNA regions (Cyt-b, 12S, and 16S genes). The results of the other three human blood stains (M1-M3) improved in relation to the last Collaborative Exercises but those related to hairs yielded a low rate of success which clearly contrasts with previous results. As a consequence of this, some labs performed additional analysis showing that the origin of this low efficiency was not the presence of inhibitors, but the low quantity of DNA present in these specific hair samples and the degradation.As a general conclusion the results emphasize the need of external proficiency testing as part of the accreditation procedure for the labs performing mtDNA analysis in forensic casework.     

Results of the GEP-ISFG collaborative study on the Y chromosome STRs GATA A10, GATA C4, GATA H4, DYS437, DYS438, DYS439, DYS460 and DYS461: population data

The Spanish and Portuguese ISFG Working Group (GEP-ISFG) carried out a collaborative exercise in order to asses the performance of two Y chromosome STR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The groups that reported correct results in all the systems were also asked to analyse a population sample in order to evaluate the informative content of these STRs in different populations. A total of 1020 males out of 13 population samples from Argentina, Brazil, Costa Rica, Macao, Mozambique, Portugal and Spain were analysed for all the loci included in the present study. Haplotype and allele frequencies of these eight Y-STRs were estimated in all samples. The lowest haplotype diversity was found in the Lara (Argentina) population (95.44%) and the highest (99.90%) in Macao (China). Pairwise haplotype analysis showed the relative homogeneity of the Iberian origin samples, in accordance with what was previously found in the European populations for other Y-STR haplotypes (http://www.ystr.org). As expected, the four non-Caucasian samples, Macao (Chinese), Mozambique (Africans), Costa Rica (Africans) and Argentina (Lara, Amerindians), show highly significant Phist values in the pairwise comparisons with all the Caucasian samples.     

Updated allelic structures of the DXS10135 and DXS10078 STR loci

DXS10135 and DXS10078 are two highly polymorphic STR loci situated in two different linkage groups on the short arm of the human X chromosome. Both loci comprise complex tetrameric repeat units which may partially explain their high degree of polymorphism. DXS10135 is relatively well characterized and is included in a commercially available kit, while DXS10078 has not been well described. We sequenced a large number of alleles of both loci to try and understand the allelic variation and as a prelude to construct allelic ladders from cloned alleles. Our data show interesting features and should encourage other workers to use these loci in forensic genetic investigations.