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PubMed2006

Two universal primer sets for species identification among vertebrates.

The aim of this study was to develop a simple method using universal primers for species identification based on direct PCR sequencing. Two primer sets were designed based on the conserved regions of the 12S and 16S rRNA loci detected by the comprehensive sequence comparison among 30 mammalian whole mitochondrial genomes. In humans, the expected sizes of PCR products of the 12S and 16S rRNAs were 215 and 244 bp, respectively. Both primer sets successfully amplified the expected PCR products from various kinds of vertebrates including mammals, birds, reptiles, amphibians, and fish, and the sequenced segments contained sufficient nucleotide differences to identify each animal species. A case example of the identification of a piece of buried bone of unknown species is presented, and the species was identified as a pig by this method.

Animals↗

PubMed2006

Application of a quasi-median network analysis for the visualization of character conflicts to a population sample of mitochondrial DNA control region sequences from southern Germany (Ulm).

Entire mtDNA control region sequences from 100 individuals in a west Eurasian population sample from southern Germany (around the city of Ulm) were generated and analyzed. The control region was amplified in one piece and sequenced with ten different sequencing primers. Sequence evaluation was performed independently. Phylogenetic analyses were used for quality assurance purposes and for the determination of the haplogroup affiliation of the samples. The sequences were scrutinized performing a quasi-median network analysis. To visualize character conflicts, frequent mutations were filtered, and the reduced data were represented by the torso of their quasi-median network. Character incompatibilities were found to be based on real biological patterns of homoplasy. The population data will be incorporated in the EMPOP database ( http://www.empop.org ).

DNA, Mitochondrial↗

PubMed2006

Development of species identification tests targeting the 16S ribosomal RNA coding region in mitochondrial DNA.

This report describes the development of a species testing system based on the diversity of nucleotide sequences in mitochondrial DNA (mtDNA) among species. Five species, human, cow, pig, dog, and cat, were considered. The partial nucleotide sequences in 16S ribosomal RNA coding region were chosen as the target for discriminating the species. The sequence diversities of this approximately 400 bp long region ranged from 15.7 to 24.1% among the five species. Sequencing analysis of this target on 50 individuals of each species (53 for dogs) revealed that the nucleotide sequences were well preserved within species. Species-specific PCR for each species was also designed, and satisfactory results with regard to both sensitivity and specificity were obtained. A validation study with DNA extracted from bovine bone exposed to the environment revealed that the PCRs designed in this study worked correctly. From the results obtained, it was shown that this testing system could be a good tool for species identification. One successful case report is also demonstrated.

Animals↗

PubMed1998

Sequence variation of a hypervariable short tandem repeat at the D1S1656 locus.

A short tandem repeat at the D1S1656 locus was sequenced in 45 selected alleles and 13 different alleles were found which were designated according to the total number of repeats. This STR is a compound hypervariable STR consisting of blocks of (TAGR) repeats with a basic sequence structure (TAGA)4(TGA)0-1(TAGA)6-16(TAGG)0-1(TG)5. The presence of a TGA, probably due to an A deletion in the fifth TAGA repeat leads to intermediate a.3 alleles. Population data showed that this is a highly polymorphic STR with a heterozygosity of more than 0.89. This fact together with its simple structure and small size (129-168 bp) makes this STR one of the most interesting DNA polymorphisms for forensic and genetic purposes.

Alleles↗

PubMed1998

Sequence variation of a variable short tandem repeat at the D18S535 locus.

A short tandem repeat in the D18S535 locus was sequenced in 25 selected alleles. A total of 8 different alleles were found which can be designated according to the total number of repeats. This STR is a simple hypervariable STR consisting of blocks of (GATA) repeats with a basic sequence structure (GATA)1(GACA)1(GATA)1 (GAT)1(GATA)9-16. Population data showed that this is a highly polymorphic STR with a heterozygosity of more than 0.80, a simple structure and small size (130-158 bp) which makes this an interesting DNA polymorphism for forensic and genetic purposes.

DNA↗

PubMed1984

Typing and subtyping of haptoglobin from native serum using disc gel electrophoresis in alkaline buffer: application to routine screening.

A method with which the six common phenotypes of human haptoglobin can be identified using unseparated serum is described. In contrast to other reported methods, both typing and subtyping of haptoglobin can be performed by polyacrylamide gel electrophoresis in alkaline buffer using 0.1-4.0 microliter of native serum with hemoglobin added. Haptoglobin-hemoglobin complexes are visualized by their peroxidase activity using benzidine and barium peroxide. This relatively inexpensive and fast method seems particularly well suited for the typing and subtyping of haptoglobin from minute amounts in large series of sera and other body fluids and thus may be useful in medical genetics and forensic medicine.

Adult↗

PubMed1987

Chromatographic separation of DNA restriction fragments.

Reversed-phase liquid chromatography (RPC-5) was the first chromatographic technique to be successfully applied to DNA restriction fragments. Size-exclusion high-performance liquid chromatography (HPLC) has the advantage of wide variability in buffer conditions but is restricted to an upper size limit at 800 base pairs. Anion-exchange HPLC is most versatile and may best be scaled up for the preparation of milligram amounts of specific DNA fragments. The preparation of the sample before and after chromatography is described. Because several applications of DNA fragments will be complemented by RNA fragments, their preparation and purification is included. Disposable small-scale columns are available for processing many samples. Future medical applications of HPLC of DNA fragments concern medical microbiology, human genetics and forensic medicine.

Animals↗

PubMed2005

Geographical heterogeneity of Y-chromosomal lineages in Norway.

Y-chromosomal variation at five biallelic markers (Tat, YAP, 12f2, SRY(10831) and 92R7) and nine multiallelic short tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385I/II and DYS388) in a Norwegian population sample are presented. The material consists of 1766 unrelated males of Norwegian origin. The geographical distribution of the population sample reflects fairly well the population distribution around the year 1942, which is the median birth year of the index persons. Seven hundred and twenty-one different Y-STR haplotypes but 726 different lineages (Y-STRs plus biallelic markers) were encountered. We observed six known (P*(xR1a), BR(xDE, J, N3, P), R1a, N3, DE, J), and one previously undescribed haplogroup (probably a subgroup within haplogroup P*(xR1a)). Four of the haplogroups (P*(xR1a), BR(xDE, J, N3, P), R1a and N3) represented about 98% of the population sample. The analysis of population pairwise differences indicates that the Norwegian Y-chromosome distribution most closely resembles those observed in Iceland, Germany, the Netherlands and Denmark. Within Norway, geographical substructuring was observed between regions and counties. The substructuring reflects to some extent the European Y-chromosome gradients, with higher frequency of P*(xR1a) in the south-west and of R1a in the east. Heterogeneity in major founder groups, geographical isolation, severe epidemics, historical trading links and population movements may have led to population stratification and have most probably contributed to the observed regional differences in distribution of haplotypes within two of the major haplogroups.

Analysis of Variance↗

PubMed2005

Population structure of Y chromosome SNP haplogroups in the United States and forensic implications for constructing Y chromosome STR databases.

A set of 61 Y chromosome single-nucleotide-polymorphisms (Y-SNPs) is typed in a sample of 2517 individuals from 38 populations to infer the geographic origins of Y chromosomes in the United States and to test for paternal admixture among African-, European-, Hispanic-, Asian-, and Native-Americans. All of the samples were previously typed with the 11 core U.S. Y chromosome short tandem repeats (Y-STRs) recommended by SWGDAM, which revealed high levels of among ethnic group variation and low levels of among-population-within-ethnic-group variation. Admixture estimates vary greatly among populations and ethnic groups. The frequencies of non-European (3.4%) and non-Asian (4.5%) Y chromosomes are generally low in European-American and Asian-American populations, respectively. The frequencies of European Y chromosomes in Native-American populations range widely (i.e., 7-89%) and follow a West to East gradient, whereas they are relatively consistent in African-American populations (26.4+/-8.9%) from different locations. The European (77.8+/-9.3%) and Native-American (13.7+/-7.4%) components of the Hispanic paternal gene pool are also relatively constant among geographic regions; however, the African contribution is much higher in the Northeast (10.5+/-6.4%) than in the Southwest (1.5+/-0.9%) or Midwest (0%). To test for the effects of inter-ethnic admixture on the structure of Y-STR diversity in the U.S., we perform subtraction analyses in which Y chromosomes inferred to be admixed by Y-SNP analysis are removed from the database and pairwise population differentiation tests are implemented on the remaining Y-STR haplotypes. Results show that low levels of heterogeneity previously observed between pairs of Hispanic-American populations disappear when African-derived chromosomes are removed from the analysis. This is not the case for an unusual sample of European-Americans from New York City when its African-derived chromosomes are removed, or for Native-American populations when European-derived chromosomes are removed. We infer that both inter-ethnic admixture and population structure in ancestral source populations may contribute to fine scale Y-STR heterogeneity within U.S. ethnic groups.

Analysis of Variance↗

PubMed2006

Nuclear and mitochondrial DNA quantification of various forensic materials.

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.

DNA Fingerprinting↗

PubMed2006

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA.

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.

Amelogenin↗

PubMed2006

LoComatioN: a software tool for the analysis of low copy number DNA profiles.

Previously, the interpretation of low copy number (LCN) STR profiles has been carried out using the biological or 'consensus' method-essentially, alleles are not reported, unless duplicated in separate PCR analyses [P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forens. Sci. Int. 112 (2000) 17-40]. The method is now widely used throughout Europe. Although a probabilistic theory was simultaneously introduced, its time-consuming complexity meant that it could not be easily applied in practice. The 'consensus' method is not as efficient as the probabilistic approach, as the former wastes information in DNA profiles. However, the theory was subsequently extended to allow for DNA mixtures and population substructure in a programmed solution by Curran et al. [J.M. Curran, P. Gill, M.R. Bill, Interpretation of repeat measurement DNA evidence allowing for multiple contributors and population substructure, Forens. Sci. Int. 148 (2005) 47-53]. In this paper, we describe an expert interpretation system (LoComatioN) which removes this computational burden, and enables application of the full probabilistic method. This is the first expert system that can be used to rapidly evaluate numerous alternative explanations in a likelihood ratio approach, greatly facilitating court evaluation of the evidence. This would not be possible with manual calculation. Finally, the Gill et al. and Curran et al. papers both rely on the ability of the user to specify two quantities: the probability of allelic drop-out, and the probability of allelic contamination ("drop-in"). In this paper, we offer some guidelines on how these quantities may be specified.

Alleles↗

PubMed2006

Species-specific oligonucleotides and multiplex PCR for forensic discrimination of two species of scallops, Placopecten magellanicus and Chlamys islandica.

Characterization of DNA that remains in seafood products after skin, scales, and shells are removed is widely used in forensic species identification, however, ordinary methods may be prohibitively expensive or time-consuming if large sample series need to be discriminated. Forensic discrimination of two species of bivalves commercially harvested from the North Atlantic, sea scallops (Placopecten magellanicus) and Icelandic scallops (Chlamys islandica), was made by means of species-specific oligonucleotides (SSOs) in a multiplex polymerase chain reaction (PCR). The test is a simultaneous in vitro amplification of a portion of the mitochondrial Cytochrome Oxidase I locus with a PCR anchor primer for a sequence identical in both species, and two alternative SSOs that selectively amplify either a 619-bp in Placopecten or a 459-bp DNA fragment in Chlamys. Fragment size and thus species identity are determined directly by gel electrophoresis. In the forensic application, analysis of more than 900 scallops from a series of samples seized from two fishing vessels showed significantly variable proportions of the species from the closed and open fisheries (Placopecten versus Chlamys, respectively). The multiplex SSO test provides a direct means of forensic identification of large population sample series, without the necessity of secondary DNA sequencing, RFLP mapping, or fingerprinting, and can be adapted to other loci and species.

Animals↗

PubMed2006

Properties of novel and widely studied Y-STR loci in three South African populations.

The objective of the present study was to examine the properties of a set of single-copy Y-STR loci to assess their suitability for forensic casework in three South African populations. Three criteria were used to select markers for assessment. Firstly, the single-copy markers of the minimal haplotype were selected based on their established use in forensic studies. Secondly, 8 markers were selected on the basis of high gene diversity values reported for several population studies, and thirdly 19 markers were chosen from a survey of Y-chromosome sequence with selections made primarily on the basis of the number of repeated elements present. Samples were typed from 101 English-speaking Caucasians, 88 Xhosa individuals and 77 Asian Indians. Gene diversity values, the number of alleles identified and the average stutter was determined for each locus.

Asian People↗

PubMed2006

Object-oriented Bayesian networks for complex forensic DNA profiling problems.

We describe a flexible computational toolkit, based on object-oriented Bayesian networks, that can be used to model and solve a wide variety of complex problems of relationship testing using DNA profiles. In particular this can account for such complicating features as missing individuals, mutation and null alleles. We illustrate the use of this toolkit with several examples, including disputed paternity with missing or additional measurements, and criminal identification. We investigate the effects on likelihood ratios of introducing mutation and/or null alleles, and show that this can be substantial even when the underlying perturbations are small.

Bayes Theorem↗

PubMed2006

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.

DNA↗

PubMed2006

Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination.

Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations.

Base Sequence↗

PubMed2006

Mobile element-based forensic genomics.

Mobile elements are commonly referred to as selfish repetitive DNA sequences. However, mobile elements represent a unique and underutilized group of molecular markers. Several of their characteristics make them ideally suited for use as tools in forensic genomic applications. These include their nature as essentially homoplasy-free characters, they are identical by descent, the ancestral state of any insertion is known to be the absence of the element, and many mobile element insertions are lineage specific. In this review, we provide an overview of mobile element biology and describe the application of certain mobile elements, especially the SINEs and other retrotransposons, to forensic genomics. These tools include quantitative species-specific DNA detection, analysis of complex biomaterials, and the inference of geographic origin of human DNA samples.

Alu Elements↗