Single Locus Probes Research Articles

Curiosity in the genes: the DNA fingerprinting story

Curiosity in the genes: the DNA fingerprinting story

Exploring Evolution in Ceboidea (Platyrrhini, Primates) by Williams-Beuren Probe (HSA 7q11.23) Chromosome Mapping

The ancestral platyrrhine karyotype was characterised by a syntenic association of human 5 and a small segment of human 7 orthologues. This large syntenic association has undergone numerous rearrangements in various phylogenetic lines. We used a locus-specific molecular cytogenetic approach to study the chromosomal evolution of the human 7q11.23 orthologous sequences (William-Beuren syndrome, WS) in various Ceboidea (Platyrrhini) species. The fluorescent in situ hybridisation of the WS probe revealed a two-way pattern of chromosomal organisation that suggests various evolutionary scenarios. The first pattern (seen in Callimico and Saimiri) includes a fairly simple disruption of the 7/5 syntenic association by a chromosome fission. The second pattern (seen in Atelinae, Alouattinae and in Callicebus) is characterised by an increasing complexity in the 7/5 association as a consequence of a series of inversions and translocations resulting in different syntenic associations. These data support recent proposals for phylogenomic groupings of New World monkeys. The study also illustrates how single-locus probe hybridisations can reveal intrachromosomal rearrangements.

A novel canine lymphoma cell line: A translational and comparative model for lymphoma research

A novel canine lymphoma cell line, OSW, was established from the malignant pleural effusion of a dog with peripheral T-cell lymphoma. The immunoprofile as determined by flow cytometry was as follows: positive for CD45, CD49d, CD18, CD11a; weakly positive for CD11b, CD11c, CD11d; and negative for CD45RA, CD1a, CD1c, CD3, TCRαβ, TCRγδ, CD4, CD5, CD8a, CD8b, CD90(Thy1), CD21, MHCII, CD14(TUK4), CD34, and MPO. Immunocytochemistry of cytospin preparations was negative for cytoplasmic CD3, CD79a, and MPO, but was positive for CD20. The cell line had an oligoclonal T-cell receptor gamma (TCRγ) gene rearrangement. Array comparative genomic hybridization (aCGH) and single locus probe (SLP) analysis showed that there were copy number increases of loci on dog chromosome 13 (CFA 13), and copy number decreases were evident for regions of CFA 11, 22, 26, 30 and 32, which include several of the more common chromosomal aberrations reported previously in canine lymphoma. The OSW cell line grows rapidly in vitro and is tumorigenic as a xenograft in SCID/NOD mice. OSW represents one of only a few reported canine lymphoma cell lines and is the most thoroughly characterized. This cell line and xenograft represent significant in vitro and in vivo models, respectively, for comparative and translational lymphoma research.

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.

Inclusiveness, effectiveness and intrusiveness: issues in the developing uses of DNA profiling in support of criminal investigations.

Current methods of forensic DNA profiling (known also as DNA fingerprinting and DNA typing), based on Polymerase Chain Reaction (PCR) amplifications of a varying number of Short Tandem Repeat (STR) loci found at different locations on the human genome, are regularly described as constituting the “gold standard for identification” in contemporary society. At a time when criminal justice systems in Europe and North America increasingly seek to utilise the epistemic authority of a variety of sciences in support of the apprehension and prosecution of suspects and offenders, genetic science and recombinant DNA technology are often singled out for particular approbation. Indeed, the development and application of DNA profiling has been widely described as the “greatest breakthrough in forensic science since fingerprinting.”Prior to the implementation of PCR based extraction and amplification methods in the 1990's, the initial uses of DNA fingerprinting (based on Multiple and Single Locus Probes) were largely confined to reactive forensic casework.

DNA analysis : Current status in court

AbstractSince the introduction of DNA profiling the methods have change considerably and so have the way that results are expressed. There have been many different techniques used to produce a DNA profile. The starting point was multilocus probes, followed by single locus probes, neither of which are now widely used, and then Short tandem Repeat (STR) analysis. Whatever the next step in DNA analysis is, the methods of interpretation will remain essentially the same. We rely on the concept and reality of the benign state for this to be used for the good of society, not the oppression of individuals or groups.

Chromosome aberrations in canine multicentric lymphomas detected with comparative genomic hybridisation and a panel of single locus probes

Recurrent chromosome aberrations are frequently observed in human neoplastic cells and often correlate with other clinical and histopathological parameters of a given tumour type. The clinical presentation, histology and biology of many canine cancers closely parallels those of human malignancies. Since humans and dogs demonstrate extensive genome homology and share the same environment, it is expected that many canine cancers will also be associated with recurrent chromosome aberrations. To investigate this, we have performed molecular cytogenetic analyses on 25 cases of canine multicentric lymphoma. Comparative genomic hybridisation analysis demonstrated between one and 12 separate regions of chromosomal gain or loss within each case, involving 32 of the 38 canine autosomes. Genomic gains were almost twice as common as losses. Gain of dog chromosome (CFA) 13 was the most common aberration observed (12 of 25 cases), followed by gain of CFA 31 (eight cases) and loss of CFA 14 (five cases). Cytogenetic and histopathological data for each case are presented, and cytogenetic similarities with human non-Hodgkin's lymphoma are discussed. We have also assembled a panel of 41 canine chromosome-specific BAC probes that may be used for accurate and efficient chromosome identification in future studies of this nature.

Paternity determination in criminal cases by DNA typing in South Ukraine

We report the results of the 1995 and 1996 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Haemogenetics. In 1995, 18 laboratories participated and in 1996, 21 laboratories participated. Each year, blood samples from three persons (child, mother and alleged father) were sent to participating laboratories which performed paternity testing according to their usual protocols. The results and answers to questionnaires concerning methods were compiled and are presented in this report. From the questionnaires, a general tendency to a more frequent use of polymerase chain reaction (PCR) based methods was seen. In 1996, 62% of the laboratories used PCR based methods. Ten per cent of the laboratories used only PCR based methods. The remaining 90% of the laboratories performed restriction fragment length polymorphism (RFLP) investigations of variable numbers of tandem repeat (VNTR) loci with single locus probes (SLPs) either alone or in combination with PCR based typing, multi locus probing, classical systems (ABO etc.), or serological HLA typing. In 1996, typing with classical systems was used in 29% of the laboratories. The majority of the laboratories performed RFLP typing of VNTR loci using very similar methods. The results and the inter-laboratory variations of the measured lengths of the DNA-fragments of the VNTR regions D2S44, D7S21, D7S22, and D12S11 of the trios were analysed. The overall coefficient of variation was 2.15% in 1995 and 1.43% in 1996. During the period 1991–1996, the inter-laboratory variation has decreased, most probably due to the fact that the methods have now been optimised and the majority of the participating laboratories have adopted the standardised method for RFLP typing with SLPs which was agreed upon for investigations in crime cases by the European DNA Profiling Group. In 1996, eight laboratories reported the results of PCR based typing of the short tandem repeat (STR) locus HumTH01, six laboratories reported results of HumVWA31A typing, and five laboratories reported the results of typing of the STR locus HumF13A1 and the VNTR locus D1S80. The results were concordant although the nomenclature was slightly inconsistent concerning the classification of an irregular repeat of the HumTH01 system.

The GEDNAP blind trial concept

The German DNA Profiling group (GEDNAP) blind trial concept, which was originally conceived as the name suggests for German laboratories only, was concipated in 1989–1990 to examine the interlaboratory variation of fragment length measurements and standardisation of the DNA single locus probes. This has now expanded and includes approximately 120 laboratories from all over Europe including Russia, and only those STR systems are now included in the blind trial which reflect the present state-of-the-art in forensic DNA testing. The success of the GEDNAP blind trial system lies in the concept of casework-oriented DNA stain testing coupled with a total participant-oriented feedback system for the presentation and evaluation of the results. This principle is also continued in the type and number of stains to be tested in subsequent trials, the selection of which is governed by the maxim of customer demand. After successful completion of the blind trial, participating laboratories are issued a certificate which is recognised in all European countries as a validation of the competence of the laboratory for forensic DNA testing. D 2003 Elsevier Science B.V. All rights reserved.

A report of the 2000 and 2001 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Genetics

During the last 10 years, the English Speaking Working Group (ESWG) of the International Society for Forensic Genetics (ISFG) has once a year arranged a Paternity Testing Workshop in which blood samples as well as a questionnaire concerning laboratory strategies were distributed to the participating laboratories. In 2000 and 2001, paper challenges were included in the workshops. Here, we present the results of the 2000 and 2001 Paternity Testing Workshops. The numbers of participating laboratories were 33 (2000) and 36 (2001). A total of 36% (2000) and 31% (2001) of the laboratories submitted typing results of variable number of tandem repeats (VNTRs) investigated with restriction fragment length polymorphism (RFLP) and single locus probes (SLPs). A total of 91% (2000) and 86% (2001) submitted typing results of polymerase chain reaction (PCR) based systems. Typing errors occurred in 0.3% of the submitted PCR-based results in 2000 and in 0.1% in 2001. The results of the paper challenges showed a high degree of variation in the formulas used for calculation of the weight of evidence of rare events such as inconsistencies or possible silent alleles. The majority of the laboratories used the same formulas for calculations of frequently occurring events.

A report of the 1997, 1998 and 1999 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Genetics

We present the results of the 1997, 1998 and 1999 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Genetics. The numbers of participating laboratories were 24 (1997), 31 (1998) and 32 (1999). In 1997, all laboratories drew the correct conclusion that the alleged father was the biological father of the child. In 1998, the alleged father was the biological brother of the child and all laboratories excluded him. The scenario in 1999 was a deficiency case consisting of mother, child and the parents of the alleged father and all but one laboratory drew the correct conclusion. The percentage of laboratories routinely performing variable number of tandem repeat (VNTR) investigations using single locus probes (SLPs) and restriction fragment length polymorphism (RFLP) decreased from 83% in 1997 to 66% in 1999. In the three workshops, more than 90% of the laboratories used polymerase chain reaction (PCR) based systems. In 1999, 80% of the laboratories performing PCR, used commercially available short tandem repeat (STR) kits. Other commonly used systems were HLA and PolyMarker investigated with PCR. Conventional systems and RFLP investigations of VNTRs with multi loci probes (MLPs) were used routinely by approximately 20% of the participating laboratories. All laboratories submitting results in the three workshops used RFLP-based VNTRs or/and PCR based VNTRs/STRs. Inter-laboratory comparisons of the results showed a very high concordance. The overall coefficients of variation between the laboratories of the results of RFLP typing of the commonly used VNTRs D2S44, D7S21, D7S22 and D12S11 were 1.2–1.3%. Consistent results were obtained in the great majority of the systems investigated by PCR and typing errors counted for less than 0.3% of the PCR based results.

Validation of short tandem repeat analysis for the investigation of cases of disputed paternity

This study details validation of two separate multiplex STR systems for use in paternity investigations. These are the Second Generation Multiplex (SGM) developed by the UK Forensic Science Service and the PowerPlex 1 multiplex commercially available from Promega Inc. (Madison, WI, USA). These multiplexes contain 12 different STR systems (two are duplicated in the two systems). Population databases from Caucasian, Asian and Afro-Caribbean populations have been compiled for all loci. In all but two of the 36 STR/ethnic group combinations, no evidence was obtained to indicate inconsistency with Hardy-Weinberg (HW) proportions. Empirical and theoretical approaches have been taken to validate these systems for paternity testing. Samples from 121 cases of disputed paternity were analysed using established Single Locus Probe (SLP) tests currently in use, and also using the two multiplex STR systems. Results of all three test systems were compared and no non-conformities in the conclusions were observed, although four examples of apparent germ line mutations in the STR systems were identified. The data was analysed to give information on expected paternity indices and exclusion rates for these STR systems. The 12 systems combined comprise a highly discriminating test suitable for paternity testing. 99.96% of non-fathers are excluded from paternity on two or more STR systems. Where no exclusion is found, Paternity Index (PI) values of > 10,000 are expected in > 96% of cases.

Population structure and dynamics of Magnaporthe grisea in the Indian Himalayas.

The population genetics of Magnaporthe grisea, the rice blast pathogen, were analyzed in a center of rice diversity (the Uttar Pradesh hills of the Indian Himalayas) using multilocus and single-, or low-copy, DNA markers. Based on DNA fingerprinting with the multilocus probe MGR586 and single-locus probes, 157 haplotypes clustered into 56 lineages (at >/=70% MGR586 band similarity, each with unique single-locus profiles) and high diversity indices were detected among 458 isolates collected from 29 sites during 1992-1995. Most valleys sampled had distinct populations (73% of the lineages were site specific) with some containing one or a few lineages, confirming the importance of clonal propagation, and others were very diverse. Widely distributed lineages suggested that migration occurs across the region and into the Indo-Gangetic plains. Repeated sampling at one site, Matli, (170 isolates, 1992-1995) yielded 19 lineages and diversity significantly greater than that reported from similar samples from Colombia and the Philippines. Analysis of allelic associations using pairwise comparisons and multilocus variance analysis failed to reject the hypothesis of gametic phase equilibrium. The Matli population shifted from highly diverse in 1992 to almost complete dominance by one lineage in 1995. Such population dynamics are consistent with recombination followed by differential survival of clonal descendants of recombinant progeny. At another site, Ranichauri, population (n = 84) composition changed from 2 to 11 lineages over 2 yr and yielded additional evidence for equilibrium. Sexually fertile and hermaphrodite isolates of both mating types were recovered from rice in both Matli and Ranichauri. We demonstrate that Himalayan M. grisea populations are diverse and dynamic and conclude that the structure of some populations may be affected to some extent by sexual recombination.

Validation of short tandem repeat analysis for the investigation of cases of disputed paternity

This study details validation of two separate multiplex STR systems for use in paternity investigations. These are the Second Generation Multiplex (SGM) developed by the UK Forensic Science Service and the PowerPlex™1 multiplex commercially available from Promega Inc. (Madison, WI, USA). These multiplexes contain 12 different STR systems (two are duplicated in the two systems). Population databases from Caucasian, Asian and Afro-Caribbean populations have been compiled for all loci. In all but two of the 36 STR/ethnic group combinations, no evidence was obtained to indicate inconsistency with Hardy-Weinberg (HW) proportions. Empirical and theoretical approaches have been taken to validate these systems for paternity testing. Samples from 121 cases of disputed paternity were analysed using established Single Locus Probe (SLP) tests currently in use, and also using the two multiplex STR systems. Results of all three test systems were compared and no non-conformities in the conclusions were observed, although four examples of apparent germ line mutations in the STR systems were identified. The data was analysed to give information on expected paternity indices and exclusion rates for these STR systems. The 12 systems combined comprise a highly discriminating test suitable for paternity testing. 99.96% of non-fathers are excluded from paternity on two or more STR systems. Where no exclusion is found, Paternity Index (PI) values of >10 000 are expected in >96% of cases.

The impact of the new fish technologies on the cytogenetics of haematological malignancies.

The impact of the new fish technologies on the cytogenetics of haematological malignancies.

Statistical methods employed in evaluation of single-locus probe results in criminal identity cases.

Evaluation of single-locus probe results in criminal-identity cases is best presented within the standard forensic framework for handling trace evidence. A comprehensive account was given by Aitken (), and the language of that text will be used here. For simplicity, suppose a stain from the scene of the crime has DNA profile type A that is known to be from the perpetrator P of the crime. Person S, who is a suspect in the crime, has profile B. In the conventional treatment, S is removed from suspicion if B ≠ A, although this matchbased approach can be avoided with a continuous treatment (). If there is a match between the stain and suspect profiles, A = B, the evidence of the match is denoted by E. There are two explanations for E: 1. C: Person S is the perpetrator P (usually the prosecution explanation). 2. C: Person S is not the perpetrator P (usually the defense explanation).

A report of the 1995 and 1996 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Haemogenetics

We report the results of the 1995 and 1996 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Haemogenetics. In 1995, 18 laboratories participated and in 1996, 21 laboratories participated. Each year, blood samples from three persons (child, mother and alleged father) were sent to participating laboratories which performed paternity testing according to their usual protocols. The results and answers to questionnaires concerning methods were compiled and are presented in this report. From the questionnaires, a general tendency to a more frequent use of polymerase chain reaction (PCR) based methods was seen. In 1996, 62% of the laboratories used PCR based methods. Ten per cent of the laboratories used only PCR based methods. The remaining 90% of the laboratories performed restriction fragment length polymorphism (RFLP) investigations of variable numbers of tandem repeat (VNTR) loci with single locus probes (SLPs) either alone or in combination with PCR based typing, multi locus probing, classical systems (ABO etc.), or serological HLA typing. In 1996, typing with classical systems was used in 29% of the laboratories. The majority of the laboratories performed RFLP typing of VNTR loci using very similar methods. The results and the inter-laboratory variations of the measured lengths of the DNA-fragments of the VNTR regions D2S44, D7S21, D7S22, and D12S11 of the trios were analysed. The overall coefficient of variation was 2.15% in 1995 and 1.43% in 1996. During the period 1991–1996, the inter-laboratory variation has decreased, most probably due to the fact that the methods have now been optimised and the majority of the participating laboratories have adopted the standardised method for RFLP typing with SLPs which was agreed upon for investigations in crime cases by the European DNA Profiling Group. In 1996, eight laboratories reported the results of PCR based typing of the short tandem repeat (STR) locus HumTH01, six laboratories reported results of HumVWA31A typing, and five laboratories reported the results of typing of the STR locus HumF13A1 and the VNTR locus D1S80. The results were concordant although the nomenclature was slightly inconsistent concerning the classification of an irregular repeat of the HumTH01 system.

Full length article

Analysis of germline mutation rate at human minisatellites among children born in areas of the Mogilev district of Belarus heavily polluted after the Chernobyl accident has been extended, both by recruiting more families from the affected region and by using five additional minisatellite probes, including multi-locus probe 33.6 and four hypervariable single-locus probes. These additional data confirmed a twofold higher mutation rate in exposed families compared with non-irradiated families from the United Kingdom. An elevated rate was seen at all three independent sets of minisatellites (detected separately by multi-locus probes 33.15, 33.6 and six single-locus probes), indicating a generalised increase in minisatellite germline mutation rate in the Belarus families. Within the Belarus cohort, mutation rate was significantly greater in families with higher parental radiation dose estimated for chronic external and internal exposure to caesium-137, consistent with radiation induction of germline mutation. The spectra of mutation seen in the unexposed and exposed families were indistinguishable, suggesting that increased mutation observed over multiple loci arises indirectly by some mechanism that enhances spontaneous minisatellite mutation.

Criticisms and concerns regarding DNA profiling

Since the first application of DNA profiling by forensic scientists to problems of identification in the late 1980's, there have been a number of aspects of such evidence which have been subject of challenge by the defence. In the UK, the most commonly used form of DNA testing at the present time is STR typing based on PCR technology, but this is relatively new, and it is the single locus probe testing which was used extensively until 1994 that is mainly drawn upon to provide examples of “criticisms and concerns”. Challenges that have been made to the various stages of DNA profiling (i.e., sample collection, production of DNA profiling, matching the profiles, evaluation of the results and presentation of evidence) are all considered. Example cases to illustrate the issues which have been raised, are presented. Some consideration is given to the more recently developed STR typing and mention is made of the guidelines laid down in the Court of Appeal in London in July 1996, concerning the way in which DNA evidence should be presented by the scientists to the Court.

DNA typing from stained sperm-positive vaginal smears: four rape cases

DNA typing from stained sperm-positive vaginal smears was performed in four cases of sexual assault. In Cases 1 and 2, a 6-month-old and a 6-week-old, Papanicolaou-stained vaginal smear with 99% propanol fixation underwent DNA analysis respectively. In Case 1, the HLA-DQα type of the sperm fraction of the smear was able to be determined following PCR, and the possibility of the suspect being the rapist could not be excluded. In Case 2, the possibility that the suspect was the rapist in the crime was excluded based on the results of DNA typing by means of two different single-locus probes. In the remaining two cases, H-E-stained smears after air drying for 4 or 5 months were the material for DNA analysis, and DNA typing was attempted by PCR-based STR system. In Case 3, there was no genetic information from sperm fraction of the smear due to the small number of spermatozoa on the glass slide. Therefore, it was impossible to determine whether the suspect was the rapist or not. In Case 4, sperm DNA from the smear was amplified and typed, and the suspect could be excluded. These four cases demonstrate that stained vaginal smears containing many spermatozoa can be used for DNA analysis in sexual assault cases.