Interrogation of short tandem repeats using fluorescent probes and melting curve analysis: a step towards rapid DNA identity screening.

Current forensic DNA profiling methods rely on the analysis of samples at specialised laboratories with an average turnaround time of several days. The ability to rapidly determine a partial profile of short tandem repeats at the point-of-arrest would be of great benefit to police forces around the world, for example enabling a suspect to be rapidly included or excluded from an investigation. We have developed a homogeneous PCR method for the interrogation of STR loci utilising fluorescent oligonucleotide probes and melting curve analysis. Alleles of the D18S51, TH01 and D8S1179 loci were differentiated and identified on the basis of target length and probe melting temperature. Assay performance was evaluated by comparing melting peak data with the AmpFlSTR SGM Plus system. The method is compatible with direct analysis of unpurified buccal swab samples, enabling a partial STR profile to be generated within 1h.

Short tandem repeat profiling provides an international reference standard for human cell lines.

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Analysis of disputed single-parent/child and sibling relationships using 16 STR loci.

This study describes the validation of short tandem repeat (STR) systems for the resolution of cases of disputed parentage where only a single parent is available for testing or where the claimed relationship of both parents is in doubt and also cases where sibship must be tested. Three separate multiplex systems the Second Generation Multiplex, Powerplex 1.2 and FFFL have been employed, giving a total of 16 STR loci. Both empirical and theoretical approaches to the validation have been adopted. Appropriate equations have been derived to calculate likelihood ratios for different relationships, incorporating a correction for subpopulation effects. An F(ST) point estimate of 1% has been applied throughout. Empirically, 101 cases of alleged father, alleged mother and child where analysed using six SLP systems and also using the three multiplex STR systems. Of the 202 relationships tested, 197 were independently resolved by both systems, providing either clear evidence of non-parentage or strong support for the relationship.

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 use of minisatellite variant repeat-polymerase chain reaction (MVR-PCR) to determine the source of saliva on a used postage stamp.

In this paper we report the identification of an individual using the MVR-PCR technique on DNA extracted from single and multiple discs (3 mm) punched from a licked stamp attached to an envelope. The individual's code was successfully and uniquely matched to one already present within a database of 500 MVR codes which had been generated in a separate laboratory. The exercise illustrates the suitability of MVR-PCR for forensic samples and demonstrates the power of this rapid and novel identification system.

Probing identity: the changing face of DNA fingerprinting.

DNA-fingerprinting technology has made a very rapid transition from being a research laboratory discovery to an applied science widely understood by, and of interest to, the general public. However, DNA fingerprinting is often portrayed as being a single generic technology, rather than a complex evolving mixture of methodologies, where specific applications demand selection of appropriate probes and techniques.

Cell line characterisation by DNA fingerprinting; a review.

DNA fingerprinting using multi-locus probes has become established as the most powerful technology for the identification and characterisation of mammalian cell lines. However, care must be taken in the technical preparation and statistical evaluation of the DNA banding patterns.

DNA fingerprinting; a biotechnology in business.

Since its discovery by Professor Alec Jeffreys, first published in 1985, DNA fingerprinting has never been far from the headlines. Cellmark, as the leading commercial DNA fingerprinting enterprise, has had the challenge of establishing its business in this high profile environment. The challenge for such a business has had three elements. Firstly and primarily to establish a laboratory system that consistently provides results to the highest standards possible. Secondly to communicate the science to its broad customer base which ranges from the general public to the experts of legal, scientific and governmental systems. Then finally there is the challenge of setting up the business in the context of the volatile, venture-capital based biotechnology market where the requirements for assured quality spar with those for cut prices. Given this backdrop it is not surprising that DNA fingerprinting has had its detractors, yet the consensus of opinion is that this technology has become a near routine business with the challenges effectively met and the success that was hoped for.

A role of DNA topoisomerases in the active dissociation of DNA minor groove-ligand complexes. A flow cytometric study of inhibitor effects.

The biological effects of a number of DNA ligands which interact with the minor groove of B-form DNA (e.g. netropsin, distamycin and Hoechst 33258) are thought to arise from the direct disturbance of the processes of DNA replication and transcription. Although ligand binding appears to be an important factor in cytotoxicity, the pathways by which drug molecules can be actively dissociated from nuclear DNA are unknown. Recent evidence suggests that minor groove ligands can distort the manner in which DNA associates with nucleosomal core particles and we have hypothesized that in an intact cell such imposed torsional stress could be subject to the action of cellular topoisomerases. We have used flow cytometry to study the effects of various inhibitors (including topoisomerase-interactive drugs) on the responses of a mutant cell line (HoeR415) to Hoechst 33342, given that the mutant shows resistance to the cytotoxicity of this DNA-specific dye due to an enhanced capacity to dissociate nuclear DNA-dye complexes. Ligand-DNA dissociation in the mutant was found to be energy-dependent but not specifically, affected by the drug-efflux blocker verapamil or by inhibitors of DNA synthesis. The topoisomerase II inhibitors novobiocin, VP16, nalidixic acid and the topoisomerase I-interactive drug camptothecin inhibited ligand-DNA dissociation to various extents with novobiocin being the most effective (100% inhibition at 1 mM). Both novobiocin and camptothecin were without effect on the nuclear loss of a DNA intercalator, adriamycin. We conclude that efficient topoisomerase activity is required for the active dissociation of DNA minor groove-ligand complexes.

The cloning of the rorB gene of Escherichia coli.

A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322. This DNA segment also complements the mitomycin C sensitivity of the rorB mutation. The gene was subcloned until defined in a fragment of 1.05 kb. Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones. Iso-electric focusing of this gene product suggests it may function in a complex.

The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation.

Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to gamma-rays but not to ultraviolet light. One new mutant of this type, named rorB, was isolated. This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN. Unlike previously reported mutants of E. coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks. The rorB gene maps close to ilvGEDAC at 84.5 min of the E. coli chromosome.

Fingerprinting cell lines: use of human hypervariable DNA probes to characterize mammalian cell cultures.

Hypervariable DNA sequences may be used as probes to derive DNA "finger-prints" for individuals. To assess the use of the human 33.15 and 33.6 probes (isolated by Jeffreys and coworkers) for characterizing cell lines of nonhuman origin, DNA from different stocks of Chinese hamster (CH) cells was screened. All CHO (ovary) sublines could be readily distinguished from CH-V79 sublines by their fingerprints, but where two stocks had been derived recently from the same line, their fingerprints were nearly identical. Similarly fingerprints of HPRT-deficient mutants derived from one cell stock were identical. A V79 x CHO fusion hybrid showed equal fingerprint band-sharing with each parent line, while early-passage diploid CH cells had a fingerprint closer to CHO than to V79. Thus these data introduce a simple means of typing cell lines to avoid cross-contamination, of checking cell hybrids, and of assessing the divergence of cell stocks from one another.

Examination of vectors with two dominant, selectable genes for DNA repair and mutation studies in mammalian cells.

A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV-gpt and HSVtk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSVtk-neo gene. The use of two-gene vectors to assess DNA repair was investigated by cutting the SV-gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types. In such experiments, selection was first applied for the undamaged HSVtk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damaged SV-gpt gene. The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts). Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct. In these experiments, some of the transformed cell colonies showed 'sectoring' on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA. For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated. Mutations of the SV-gpt gene in these cells were isolated while maintaining selection for the HSVtk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events. Spontaneous and X-ray-induced mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence. Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E. coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.

Vector-mediated DNA double-strand break repair analysis in normal, and radiation-sensitive, Chinese hamster V79 cells.

DNA double-strand break repair was assessed in 2 new radiation-sensitive V79 hamster cell lines (irs1 and irs2) by their ability to rejoin restriction endonuclease cuts in a transferred selectable SV40--E. coli gpt recombinant gene. The studied gene was carried in the vector pPMH16 which also contained a second selectable HSVtk-neo recombinant gene which acted as a control for DNA transformation. The parental V79 cells showed correct rejoining of KpnI and EcoRV double-strand breaks in approximately 18% and 36% of transformants respectively (correcting for the expression of undamaged gpt in neo+ transformants). irs1 shows a significantly reduced (approximately 3-fold) ability to rejoin correctly such double-strand scissions. However, irs2 rejoined such lesions as correctly as the V79 cells. The data are discussed in the context of the assay and the possible repair deficiencies of these radiosensitive mutant cells.

A mammalian cell mutant with enhanced capacity to dissociate a bis-benzimidazole dye-DNA complex.

The bis-benzimidazole dyes (specifically Hoechst 33258 and the more lipophilic derivative Hoechst 33342) are non-intercalating AT base pair-specific ligands which bind to cellular DNA by non-covalent association with the minor groove. The interaction of dye with cellular DNA is thought to be the principal pathway for the cytotoxic, mutagenic and DNA-damaging properties of such agents. Upon binding and near UV light excitation, dye molecules exhibit fluorescence enhancement such that dye/DNA association and dissociation in individual cells can be monitored by flow cytometry. We have studied dye uptake and the DNA--dye dissociation characteristics of a Hoechst 33258-resistant mouse cell line (HoeR415) compared to the response of the parental cell line Ltk-. HoeR415 was found to show similar levels of cross resistance (approximately 10-fold) to Hoechst 33258 and Hoechst 33342 compared to parental responses except that the more lipophilic ligand was approximately 30-fold more toxic. Estimates of Hoechst 33342 uptake using flow cytometry or radiolabelling methods indicated that resistance could not be attributed to reduced cellular uptake, low initial levels or different modes of DNA binding. Both cell lines showed similar initial levels of dye-induced DNA strand-breakage. However, Hoechst 33342 resistance did correlate with an enhanced capacity (10-fold) of HoeR415 to remove dye from cellular DNA compared with the relatively long retention (T 1/2 300 min) of ligand by the parental cell line. Our results are consistent with the view that ligand persistence rather than indirect DNA damage is a more important factor in the cytotoxicity of non-intercalating DNA-binding ligands. A model is presented of the cellular processes of DNA damage recognition and surveillance for ligands which interact with the minor groove of DNA.

Molecular studies on the nature of the repair defect in ataxia-telangiectasia and their implications for cellular radiobiology.

We have utilized DNA transfer and recombinant DNA techniques to probe DNA double-strand break repair in the human ionizing radiation-sensitive genetic syndrome ataxia-telangiectasia (A-T). Using restriction enzyme-generated double-strand breaks in the coding sequence of a selectable gene we have detected a significantly greater frequency of mis-repair of such breaks in a permanent A-T cell line compared with cell lines of normal radiosensitivity. This mis-repair in A-T can plausibly explain many of the clinical features of the disease but was insufficiently detailed to address the broad problem of DNA repair mechanisms relevant to ionizing radiation-induced damage. To extend these observations of DNA double-strand break mis-repair we have now applied this type of repair assay to novel, de novo induced mammalian X-ray-sensitive cell lines and to appropriate Escherichia coli mutants. In both cellular systems we have now found some equivalence to the A-T repair defect. In particular, studies on one E. coli mutant have provided evidence suggesting an involvement of a topoisomerase activity in DNA double-strand break mis-repair, which may be relevant to the biochemical defect in A-T.