Paul Debenham

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.

Abstract 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 FST 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.

Rapid typing of STRs in the human genome by HyBeacon melting.

A new method based on DNA melting has been developed for the rapid analysis of STRs in the human genome. The system is based on homogeneous PCR followed by fluorescence melting analysis and utilises a HyBeacon probe combined with a PCR primer-blocker oligonucleotide. The use of blockers of different length permits identification of the full range of common D16S539 repeats enabling detection of 99.8% of known alleles. The interrogation of STRs can be carried out on standard genetic analysis platforms and could be applied to other loci to form the basis of a bespoke high-throughput system for use in forensic analysis, particularly as fluorescent genetic analysis platforms are now available for high-resolution melting. This methodology may be suitable for rapid forensic DNA analysis at the point-of-arrest or in a custody suite where it is important to identify an individual from a small group of suspects/detainees.

Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNP gene using linear fluorescent probes and melting curve analysis

BackgroundResistance and susceptibility to scrapie has been associated with single nucleotide polymorphisms located within codons 136, 154 and 171 of the ovine prion protein gene (PRNP). Dual-labelled HyBeacon probes were developed to analyse single and clustered polymorphisms within these and neighbouring codons.MethodsExtracted DNAs and unpurified blood samples were genotyped with respect to polymorphisms in PRNP codons 136, 141, 154 and 171. PCR amplicons were investigated using a LightTyper instrument, measuring the stability of probe/target hybridisation through peak melting temperatures and determining the sequence of nucleotides at polymorphic sites.ResultsThe performance of HyBeacon assays was evaluated in a validation study comparing genotypes with those obtained using a primer extension assay (Sequenom MassEXTEND) analysed on a MALDI-ToF mass spectrometer. Over 12,000 sheep samples were successfully genotyped, reliably detecting A136, V136, T136, T137, L141, F141 R154, H154, L168, R171, Q171, H171 and K171 sequence variants using only 4 HyBeacon probes.ConclusionHyBeacon assays provide an extremely robust and accurate method for the analysis of single and clustered PRNP polymorphisms in a high-throughput format. The flexibility of the diagnostic tests ensures that samples are correctly genotyped even in the presence of additional sequence variations that flank the polymorphisms of interest. Such sequence variations may also be neutralised using universal bases such as 5-nitroindole if required.

Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry

Mitochondrial DNA is commonly used in identity testing for the analysis of old or degraded samples or to give evidence of familial links. The Abbott T5000 mass spectrometry platform provides an alternative to the more commonly used Sanger sequencing for the analysis of human mitochondrial DNA. The robustness of the T5000 system has previously been demonstrated using DNA extracted from volunteer buccal swabs but the system has not been tested using more challenging sample types. For mass spectrometry to be considered as a valid alternative to Sanger sequencing it must also be demonstrated to be suitable for use with more limiting sample types such as old teeth, bone fragments, and hair shafts. In 2009 the Commonwealth War Graves Commission launched a project to identify the remains of 250 World War I soldiers discovered in a mass grave in Fromelles, France. This study characterises the performance of both Sanger sequencing and the T5000 platform for the analysis of the mitochondrial DNA extracted from 225 of these remains, both in terms of the ability to amplify and characterise DNA regions of interest and the relative information content and ease-of-use associated with each method.

HyBeacon probes for rapid DNA sequence detection and allele discrimination.

HyBeacon probes are single-stranded oligonucleotides with one or more internal base(s) labeled with a fluorescent dye. When a probe forms a duplex with its target sequence, the level of fluorescence emission increases considerably. HyBeacons have been developed as new tools for rapid sequence detection and discrimination and have been employed in a wide variety of applications including infectious diagnostics and analysis of human polymorphisms. Single-labeled (FVG1) and dual-labeled (FVG11) probes were designed to analyze the factor V Leiden (R506Q) polymorphism which causes an increased risk of deep vein thrombosis and pulmonary embolism. Detection and identification of factor V alleles is performed by melting curve analysis and determination of probe melting temperature (T(m)). HyBeacon hybridization to the glutamine allele (Q) causes the formation of mismatched DNA duplexes that are detected through decreases in T(m). HyBeacon probes are included in homogeneous PCR assays to genotype samples with respect to the factor V polymorphism within 20 min, using purified DNAs and unpurified saliva/blood samples. This paper describes the preparation of homogeneous PCR assays, LightCycler target amplification, and subsequent melting curve analysis. This chapter also describes the use of homologous oligonucleotides and melting curve analysis as a method for probe evaluation.

Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes – a homogenous system for sequence-specific detection

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.

Synthesis and use of universal sequence probes in fluorogenic multi-strand hybridisation complexes for economical nucleic acid testing.

Analysis of nucleic acid amplification products has become the gold standard for applications such as pathogen detection and characterisation of single nucleotide polymorphisms and short tandem repeat sequences. The development of real-time PCR and melting curve analysis using fluorescent probes has simplified nucleic acid analyses. However, the cost of probe synthesis can be prohibitive when developing large panels of tests. We describe an economic two-stage method for probe synthesis, and a new method for nucleic acid sequence analysis which together considerably reduce costs. The analysis method utilises three-strand and four-strand hybridisation complexes for the detection and identification of nucleic acid target sequences by real-time PCR and fluorescence melting.

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.