Kevin Sullivan

The application of mitochondrial DNA typing to the study of white Caucasian genetic identification

SummaryMitochondrial DNA (mtDNA) from 100 unrelated British White Caucasians was extracted, amplified and directly sequenced. Sequences of approximately 800 nucleotides were obtained from 2 hypervariable segments within the non-coding region of the mitochondrial genome. A total of 91 different sequences were observed with an average nucleotide diversity of 1.1%. The most diverse pair of sequences differed at 3.6% of their nucleotide (nt) sites. Comparison to a consensus reference sequence showed that each region was polymorphic to a similar extent. Different methods of genetic analysis were used to examine the variation in each region, including pairwise comparisons, which demonstrated that although the data did not fit a Poisson distribution, the fit was closer to a Negative Binomial distribution.ZusammenfassungDie mitochondriale DNA (mtDNA) von 100 unverwandten britischen Kaukasiern wurde extrahiert, amplifiziert und direkt sequenziert. Die Sequenzen von ungefähr 800 Nucleotiden wurden von 2 hypervariablen Segmenten innerhalb der nicht-kodierenden Region des mitochondrialen Genoms erhalten. Insgesamt wurden 91 unterschiedliche Sequenzen beobachtet mit einer durchschnittlichen Nukleotid-Diversifität von 1,1 %. Die meisten unterschiedlichen Sequenz-Paare differierten an 3,6% ihrer Nukleotid-(nt)-Positionen. Ein Vergleich mit einer Konsensus-Sequenz zeigte, daß jede Region bis zu einem ähnlichen Ausmaß polymorph war. Unterschiedliche Methoden der genetischen Analyse wurden angewandt, um die Variation in jeder Region zu untersuchen, unter Einschluß paarweiser Vergleiche. Diese Untersuchung zeigte, daß die Daten nicht einer Poisson-Verteilung entsprachen, sie paßten eher zu einer negativen Binomial-Verteilung.

Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin

SummaryGender identification of forensic samples was determined by amplifying a segment of the X-Y homologous gene amelogenin. Using a single pair of primers spanning part of the first intron, 106 by and 112 by PCR products were generated from the X and Y homologues respectively, which were then resolved by agarose gel electrophoresis. This test enabled as little as 20 pg of DNA from severely degraded bones to be amplified and typed in a single tube reaction. Furthermore, using dye-labelled primers, it was possible to quantitate, by automated fluorescence detection, the relative yields of X and Y -specific PCR products generated from mixtures of male and female DNA. The versatility of this sex test was further demonstrated by co-amplifying with the HLA-DQA1 Amplitype kit in a combined gender/identity DNA test.ZusammenfassungDurch Amplifikation eines Segments des X-Y-homologen Gens Amelogenin wurde eine Geschlechtsidentifikation forensischer Proben durchgeführt. Mit einem einzigen Primerpaar, welches einen Teil des ersten Introns überspannt, wurden PCR-Produkte mit 106bp und 112bp von den homologen Anteilen des X- und des Y-Chromosoms generiert, welche dann mit Hilfe der Agarosegelelektrophorese aufgetrennt wurden. Dieser Test erlaubte es, daß so geringe Mengen wie 20pg DNA von stärkergradig degradierten Knochen amplifiziert und ineiner Einzelreaktion typisiert wurden. Durch Benutzung von farbstoffmarkierten Primern war es ferner möglich, durch automatisierte Fluoreszenzdetektion die relative Ausbeute von X- und Yspezifischen PCR-Produkten zu quantifizieren, wie sie von Mischungen männlicher und weiblicher DNA generiert wurden. Die Vielseitigkeit dieses Sex-Tests wurde ferner dadurch nachgewiesen, daß eine Co-Amplifikation mit dem HLA-DQA1 „Amplitype”-Kit in einem kombinierten Geschlechtsbestimmungs-und Identifizierungs-DNA-Test möglich war.

Identification of human remains by amplification and automated sequencing of mitochondrial DNA

SummaryThe highly decomposed remains of a corpse were identified by the amplification and direct sequencing of mitochondrial (mt) DNA. Degraded DNA was extracted from bone fragments and a necrotic skin sample and amplified at 2 hypervariable segments within the mitochondrial non-coding region using 2 rounds of nested PCR. Both strands of the amplified regions were sequenced and compared with each other to ensure fidelity of the data. Sequences from the bone and skin were found to be identical and matched data generated from a blood sample provided by a presumptive sister of the deceased. Thus, this evidence was consistent with the sister and the deceased being related.ZusammenfassungDie hochgradig zersetzten Überbleibsel einer Leiche wurden mit Hilfe der Amplifikation und der direkten Sequenzierung der mitochondrialen DNA identifiziert. Degradierte DNA wurde von Knochenfragmenten und von einer nekrotischen Hautprobe extrahiert und in zwei hypervariablen Segmenten der mitochondrialen nicht-kodierenden Region amplifiziert unter Anwendung zweier Runden von „nested” PCR. Beiden Stränge der amplifizierten Regionen wurden sequenziert und miteinander verglichen, um die Vertrauenswürdigkeit der Daten zu sichern. Die Sequenzen von Knochen und von der Haut waren identisch und paßten zu den Daten aus einer Blutprobe, welche von einer mutmaßlichen Schwester des Verstorbenen herrührte. Dieser Beweis war somit konsistent mit der Annahme, daß Schwester und Verstorbene verwandt waren.

The analysis of hypervariable DNA profiles: problems associated with the objective determination of the probability of a match

SummaryA computerised system has been used to store DNA profiles from 3 hypervariable loci. This initial survey illustrates that band matching is only possible after analysis of the errors associated with electrophoretic systems. A number of databases have been constructed with the three probes investigated and two methods of frequency determination, ‘binning’ and ‘sliding window’ fitting, have been compared.

DNA typing from human faeces

A method has been developed for the forensic analysis of faeces by DNA amplification and direct sequencing of a polymorphic segment of mitochondrial DNA. Starting from as little as 10 mg wet weight of faeces, DNA was extracted by a variety of protocols and amplified using primers specific to hypervariable region I of the mitochondrial control region. The resulting amplification products were sequenced in solid phase using an automated DNA sequencer. In total, mtDNA sequences were generated from the faeces of nine Caucasians and compared with sequences generated from their respective blood samples. Sequences of faeces and blood samples from the same individual were identical in every case, but a range of 1–10 nucleotide differences was observed between individuals, with an average sequence variation of approximately 4.88 per 400 bp. Of the various extraction protocols assessed in this study, greatest success rates were achieved using magnetisable beads to bind and purify the DNA. STR analysis of DNA extracted from faeces was not routinely possible.

Analysis of 6 VNTR loci by ‘multiplex’ PCR and automated fluorescent detection

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to ‘allelic dropout’ was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.

Characterisation of HLA DQα for forensic purposes. Allele and genotype frequencies in British Caucasian, Afro-Caribbean and Asian populations

SummaryAllele and genotype frequencies for British Caucasian, Afro-Caribbean and Asian populations were determined for a total of over 600 unrelated individuals at the HLA-DQα locus. These were analysed by polymerase chain reaction amplification of the DNA followed by hybridisation to allele specific oligonucleotide probes in a reversed dot-blot test. Six different alleles were detected and the allele distributions for the 3 populations analysed displayed significant differences. However, the British Caucasian genotypes were statistically very similar to previously published data from US Caucasians as were British Afro-Caribbean genotype frequencies with US Black data. In Caucasians the allele frequencies ranged from 5.2% to 26.9% with a power of discrimination of 0.93. DQα genotype frequencies of Caucasian and Afro-Caribbean populations do not deviate from Hardy-Weinberg equilibrium. However, the Asian data displayed significant deviation due to excess homozygotes.ZusammenfassungDie Allel- und Genotyp-Frequenzen am HLA-DQα Locus wurden für britische Europäder, Afro-Karibier und für asiatische Bevölkerungen an mehr als 600 unverwandten Personen bestimmt. Die Bestimmung erfolgte durch Amplifikation der DNA mit Hilfe der Polymerase-Kettenreaktion, gefolgt von einer Hybridisierung mit allelspezifischen Oligonucleotid-Sonden in einem umgekehrten „Dot-Blot”-Test. Sechs verschiedene Allele wurden nachgewiesen, und die Allel-Verteilungen für die drei untersuchten Bevölkerungen läßt signifikante Unterschiede erkennen. Jedoch waren die britisch-europdischen Genotypen statistisch sehr ähnlich zu früher veröffentlichten Daten von den US-Europädern, wie auch die britischen afrokaribischen GenotypFrequenzen zu den Daten der US-Schwarzen sehr ähnlich sind. Bei Europäern variierten die Allel-Frequenzen von 5,2% bis 26,9%, die Diskriminationsmöglichkeit betrdgt 0,93. Die DQα Genotyp-Frequenzen für europäi-sche und afrokaribische Populationen weichen nicht vom Hardy-Weinberg-Gleichgewicht ab. Jedoch zeigen die asiatischen Daten eine signifikante Abweichung, welche durch einen Überschuß an Homozygoten bedingt ist.

DNA analysis in the case of Kaspar Hauser

Abstract In 1828 a mysterious young man appeared in Nürnberg, Germany, who was barely able to speak or walk but could write down his name, Kaspar Hauser. He quickly became the centre of social interest but also the victim of intrigue. His appearance, his origin and assassination in 1833 were, and still are, the source of much debate. The most widely accepted theory postulates that Kaspar Hauser was the son of Grand Duke Carl von Baden and his wife Stephanie de Beauharnais, an adopted daughter of Napoleon Bonaparte. To check this theory, DNA analysis was performed on the clothes most likely worn by Kaspar Hauser when he was stabbed on December 14th, 1833. A suitable bloodstain from the underpants was divided and analysed independently by the Institute of Legal Medicine, University of Munich (ILM) and the Forensic Science Service Laboratory, Birmingham (FSS). Mitochondrial DNA (mtDNA) was sequenced from the bloodstain and from blood samples obtained from two living maternal relatives of Stephanie de Beauharnais. The sequence from the bloodstained clothing differed from the sequence found in both reference blood samples at seven confirmed positions. This proves that the bloodstain does not originate from a son of Stephanie de Beauharnais. Thus, it is becoming clear that Kaspar Hauser was not the Prince of Baden.

Cloning and Characterisation of Novel Single Locus Probes for Forensic Purposes

From the analysis of multi-locus probes (Jeffreys et al, 1986; Fowler et al, 1988) it has previously been demonstrated that there are numerous hypervariable loci in the human genome. Many hypervariable loci have been fortuitously identified (Wyman and White, 1980; Bell et al, 1982; Higgs et al, 1981; Capon et al, 1983). A cloning strategy, used specifically to isolate hypervariable DNA was developed by Wong et al (1986, 1987) who used lambda libraries of human DNA; the insert consisting of size selected high molecular weight (5–15kB) DNA fragments. This procedure enriched the library for tandemly repeated minisatellites. Recently, Armour et al (1990) introduced a more successful and simpler method for cloning hypervariable DNA by constructing Charomid libraries (Saito and Stark, 1986). Charomids are cosmid derived vectors which do not have the same size constraints of lambda vectors, hence the cloning of rather unstable minisatellite fragments, which tend to lose repeats, is much improved.

Response to low level DNA profiling

Dear Sir/Madam, DNA reviews: low level DNA profiling For. Sci. Med. Pathol. (2008) 4: 129-131. E. A. M. Graham. (1) The low copy number (LCN) technique as used by the Forensic Science Service (FSS) has been used in casework since 1999. The FSS has always engaged in a rigorous programme of research, development, and validation of any DNA profiling technique it uses which supplies DNA profile results to the Criminal Justice System (CJS). The LCN DNA profile technique was no exception to this process and its implementation into forensic casework followed the same process as other previous DNA profiling tests. Nevertheless in 2006 the validation of the technique came under scrutiny in the case of R-v-Hoey in relation to the Omagh bomb enquiry and other linked explosive devices in Northern Ireland. Even prior to the verdict and the judgement in this case, the Home Office had ordered an independent review of the science underlying the LCN approach. On April 11, 2008, the office of the UK Forensic Regulator (a department of the Home Office) issued a detailed independent review of the adequacy of the DNA LCN technique, which was produced by Professor Brian Caddy, Graham R. Taylor, and Dr Adrian M. T. Linacre. This review reported that the science supporting the delivery of low template DNA (LtDNA) analysis was sound, and that the three companies providing this service to the Criminal Justice System (the Forensic Science Service Ltd, LGC Forensics, and Orchid Cellmark Ltd) have validated their processes in accord with accepted scientific principles using both 28 (standard) and 34 (LCN) PCR cycles for extracts containing less than 200 pg of DNA. On May 7, 2008 the Forensic Regulator, Andrew Rennison, published a response to this review. The Regulator concludes: