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.

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.

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.

An investigation of the robustness of the consensus method of interpreting low-template DNA profiles.

Forensic STR profiles generated from low-template DNA samples are more noticeably subject to effects such as allele dropout, contamination with spurious alleles ('drop-in') and proportionally larger stutter. The profiles obtained are frequently partial, and are challenging to interpret. Specifically, interpretation guidelines which are used when the template DNA is in the optimal range for the STR test kit in use must be adapted to the low-template regime. A commonly used approach to such modified interpretation is known as the 'consensus' or 'biological' method, and relies on replication to achieve reliable results. We have carried out a study to assess the robustness of the consensus method as applied to SGM Plus(®) STR profiles obtained after applying a set of post-PCR purification methods together known as DNA SenCE, and report the results here. Multiple repeat analysis of DNA at five template quantities (ranging between 5pg and 100pg) and from five single donors, was carried out, and the resulting profiles were used to produce consensus profiles using several different evaluation criteria. Our aim was to determine whether the consensus profiles produced are conservative, that is, that the alleles reported are associated with the donor and that drop-in is reduced or eliminated. To this end, the alleles in the consensus profiles were compared with those of the donors, and the degree of concordance determined. The results suggest that increasingly stringent requirements for the number of times an allele must be observed in a set of repeat runs do, as expected, reduce the effect of drop-in, but also reduce the evidential value of the consensus profiles. However, the former is reduced to a much greater extent than the latter, resulting in a relative increase in profile information content versus drop-in peak risk with increased stringency. We also found that approximately half of the non-donor peaks appearing in consensus profiles were in -4 stutter positions for donor alleles present in the same profile, suggesting that many of these so-called drop-in alleles are, in fact, large stutter peaks rather than 'true' drop-in. Nevertheless, the appearance of non-donor peaks in a profile, including what are assumed to be oversized stutter peaks, appears to be an essentially random event.

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.

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.

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

BACKGROUND: Resistance 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. METHODS: Extracted 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. RESULTS: The 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. CONCLUSION: HyBeacon 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.

Genetics of epilepsy: epilepsy research foundation workshop report.

The Sixth Epilepsy Research Foundation workshop, held in Oxford in March 2006, brought together basic scientists, geneticists, epidemiologists, statisticians, pharmacologists and clinicians to consider progress, issues and strategies for harnessing genetics to improve the understanding and treatment of the epilepsies. General principles were considered, including the fundamental importance of clear study design, adequate patient numbers, defi ned phenotypes, robust statistical data handling, and follow-up of genetic discoveries. Topics where some progress had been made were considered including chromosomal abnormalities, neurodevelopment, hippocampal sclerosis, juvenile myoclonic epilepsy, focal cortical dysplasia and pharmacogenetics. The ethical aspects of epilepsy genetics were reviewed. Principles and limitations of collaboration were discussed. Presentations and their matched discussions are produced here. There was optimism that further genetic research in epilepsy was not only feasible, but might lead to improvements in the lives of people with epilepsy.

Imagery rehearsal in the treatment of posttraumatic nightmares in Australian veterans with chronic combat-related PTSD: 12-month follow-up data.

Nightmares are often a distressing symptom for veterans with chronic combat-related posttraumatic stress disorder (PTSD). A psychological treatment that has recently shown considerable promise is Imagery Rehearsal Therapy (IRT). In a pilot study by the current authors, IRT was demonstrated to be effective in the treatment of posttraumatic nightmares in a group of combat veterans up to 3-month posttreatment. This study reports the 12-month follow-up data of the pilot study, examining the longer term outcome of the IRT treatment. Twelve Australian Vietnam veterans with chronic combat-related PTSD were treated with 6 once weekly sessions of imagery rehearsal and assessed using standardised measures of nightmare frequency and intensity, PTSD, depression, anxiety and broader symptomatology at intake, posttreatment, and 3-and 12-month follow-up. Significant improvements in targeted nightmare frequency and intensity were evident to 12-month posttreatment. Similarly, improvements in overall PTSD, depression, anxiety, and broader based symptomatology were also maintained to 12 months. This study provides preliminary evidence that the positive treatment effects of IRT on posttraumatic nightmares, PTSD, and broader symptomatology in males with chronic combat-related PTSD are maintained in the longer term.

MMPI-2 based subgroups of veterans with combat-related PTSD: differential patterns of symptom change after treatment.

Considerable research has focused on the use of the MMPI to assess posttraumatic stress disorder (PTSD) through identification of mean profile configurations and the development of PTSD subscales. Little work, however, has addressed the heterogeneity of profiles evident in PTSD populations. This study investigated the MMPI-2 profiles of 158 Australian treatment-seeking Vietnam veterans with combat-related PTSD to identify distinct subgroups. Three robust subgroups were identified on the basis of their MMPI-2 profile and compared on PTSD and associated symptomatology. These subgroups consisted of a mild PTSD group with subclinical personality pathology, and two severe PTSD groups that differed in levels of personality disturbance and general psychopathology. Most notably, differences between these latter two groups occurred in the areas of externalization, alienation, and propensity for acting out. These groups were labeled as subclinical, trauma profile, and global. The groups demonstrated significant differences in the patterns of recovery after treatment. The subclinical group demonstrated little change after treatment. In contrast, the trauma profile and global groups both improved, although the trauma profile group demonstrated greater PTSD symptom reduction than the global group.

MMPI-2 as a predictor of change in PTSD symptom clusters: a further analysis of the Forbes et al. (2002) data set.

In this study, we reanalyzed the Forbes et al. (2002) data set to examine the Minnesota Multiphasic Personality Inventory (MMPI-2; Butcher, Dahlstrom, Graham, Tellegen, & Kaemmer, 1989) as a differential predictor of change across posttraumatic stress disorder symptom clusters following treatment in 141 Vietnam veterans. A series of partial correlation and linear multivariate regression analyses, controlling for initial symptom severity, identified several scales predictive of symptom change. None of the MMPI-2 scales, however, emerged as predictors of change in reexperiencing symptoms. Social alienation and marital distress were the most potent predictors for avoidance symptoms. Anger, alcohol use, and hypomania were the most potent predictors for the hyperarousal symptoms. Of the personality disorders, borderline personality was the strongest predictor of change in the avoidance and hyperarousal clusters. Further replication of the findings of this article and those reported by Forbes et al. (2002) is required.

The MMPI-2 as a predictor of symptom change following treatment for posttraumatic stress disorder.

This study sought to examine the impact of personality factors on symptom change following treatment for 141 Vietnam veterans with chronic combat-related posttraumatic stress disorder (PTSD) using the Minnesota Multiphasic Personality Inventory-2 (Butcher, Dahlstrom, Graham, Tellegen, & Kaemmer, 1989). A series of partial correlation and linear multivariate regression analyses identified social alienation, associated with anger and substance use, as the most potent negative predictor of symptom change. Of the scales assessing personality disorder, Borderline Personality was identified as the strongest negative predictor of outcome. Regression analyses examining the most salient scales identified 5 items that contributed 14% of the variance in the prediction of change scores independently of the 21% accounted for by pretreatment PTSD severity.