Browsing by Author "Larcombe, Lee"

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    Development of an automated identification system for nanocrystal encoded microspheres in flow cytometry
    (Cranfield University, 2009-04) Gallagher, Clair; Cullen, David C.; Larcombe, Lee
    Aim: This work sets out to use haplotype-based tagSNP selection and a systematic in silico analysis for design of multiplex-compatible PCR primer and SAT probe sets to capture maximum variation with minimum tests across candidate genes IGF1, IGFBP1 and IGFBP3. Additionally, the work aims to develop a number of robust, high-efficiency, high-specificity multiplex PCR constructs for amplification of these targets and to demonstrate the applicability of these target types to suspension array genotyping for non-insulin-dependant diabetes mellitus association facilitation. Methods: Haplotypes for predominantly European Caucasian populations were constructed and tagSNP selection performed using Haploview to capture maximum variation across candidate genes IGF1, IGFBP1 and IGFBP3. Extensive in silico analysis was performed for design, evaluation and selection of robust high-specificity primer and probe pairs, suitable for downstream multiplex PCR and SAT analysis. Singleplex endpoint and real-time PCR was performed for primer pair profile determination which informed multiplex PCR set construction and optimisation. The applicability of this complex target type to suspension array-based genotyping was investigated using a model probe pair using both quantum dot-encoded and fluorophore-encoded microspheres. Results: Haploview was used for haplotype construction and linkage disequilibriumbased tagSNP selection across candidate genes, reducing the number of SNP targets from 292 to 32 with minimal information loss. Extensive evaluation of potential tagSNPs was performed and 29 SNPs, representing 29 bins across target genes were designed for multiplex analysis. Singleplex end-point and real-time PCR was performed for primer pair profile determination which allowed four multiplex PCR sets to be constructed and optimised for simultaneous amplification of 14, six, five and two targets. The applicability of this complex target type (14-plex) to suspension array-based genotyping was demonstrated using a model probe pair. Conclusion: In silico analysis techniques have been applied for successful development of four robust multiplex PCR sets (14-plex, 6-plex, 5-plex and 2-plex) which display high-efficiency and target-specific amplification of tagSNPs, capturing maximum assaycompatible variation across candidate genes IGF1, IGFBP1 and IGFBP3 for European Caucasian populations. The applicability of these multiplex PCR constructs to suspension array-based genotyping has been demonstrated, thus paving the way for development of large multiplex suspension array-based genotyping assays using probes designed during the course of this work. This work offers the potential for comprehensive association analyses to become more accessible to the wider-scientific community by facilitating reduced genotyping burdens which allow increased accessibility for powerful association.
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    The effect of oxidation on the stability of G-quadruplex DNA : implications for oncogene expression
    (Cranfield University, 2011-10) Stebbeds, William Joshua David; Larcombe, Lee; Lunec, Joseph
    G-quadruplexes (G4-DNA) are a class of secondary structures formed from Guanine rich sequences. In recent years these structures have been implicated in both telomere maintenance and oncogene expression, and have been shown to be abundant in upstream promoter regions and at telomeric ends. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. The oxidation of deoxyguanosine to 8-oxo-2’deoxyguanosine (8-oxo-dG) is the most common result when DNA is under oxidative stress and as such, the G-rich sequences that form G-quadruplexes can be viewed as potential “hot-spots” for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This project used both in silico and in vitro methods to observe the effect of oxidation on the G-quadruplex structure and the consequences in oncogene expression, using two biologically relevant G-quadruplex structures, those found in the promoter regions of the proto-oncogenes c-Myc and c-Kit as proof of concept. Molecular dynamics (MD) simulations were performed (isothermic, isobaric 500ns unrestrained simulation in explicit solvent and counterions) on the c-Kit and c-Myc G-quadruplex structures with and without 8-oxo-dG incorporated into the central tetrad. FRET experiments were performed on these same structures, observing the conformation of sequences known to form G-quadruplexes under near physiological conditions and subjected to oxidative stress, through Fenton chemistry. Gene expression data analyses were also performed to evaluate the prevalence of different G-quadruplex forming motifs (GQMs) in genes affected by oxidation.Although no relevant information was gained from the FRET experiments, the MD results constitute the longest simulations of this type performed on the c-Myc and c-Kit G-quadruplex structures published to date and predict the high stability of these structures under normal physiological conditions. They also clearly demonstrate a destabilising effect of oxidation on G-quadruplex structures, with the extent of the effect dependent on the structure oxidised. Furthermore, gene expression data analysis showed that genes whose expression is significantly altered when subjected to oxidative stress are statisticallymore likely to contain a GQM than the remainder of the genome, through the use of significance testing. These findings demonstrate a differential effect of oxidation on G-quadruplexes, likely dependent on other known characteristics affecting G4 stability such as loop length and sequence. Results also point towards this mechanism affecting gene expression. This is suggestive of a novel route for oxidation mediated carcinogenesis, through upregulation of oncogene expression or possibly downregulation of tumour suppression genes.
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    Meta-analysis of expression of l(3)mbt tumor-associated germline genes supports the model that a soma-to-germline transition is a hallmark of human cancers
    (John Wiley & Sons, Ltd, 2014-05-15T00:00:00Z) Feichtinger, Julia; Larcombe, Lee; McFarlane, Ramsay J.
    Evidence is starting to emerge indicating that tumorigenesis in metazoans involves a soma-to-germline transition, which may contribute to the acquisition of neoplastic characteristics. Here, we have meta-analyzed gene expression profiles of the human orthologs of Drosophila melanogaster germline genes that are ectopically expressed in l(3)mbt brain tumors using gene expression datasets derived from a large cohort of human tumors. We find these germline genes, some of which drive oncogenesis in D. melanogaster, are similarly ectopically activated in a wide range of human cancers. Some of these genes normally have expression restricted to the germline, making them of particular clinical interest. Importantly, these analyses provide additional support to the emerging model that proposes a soma-to-germline transition is a general hallmark of a wide range of human tumors. This has implications for our understanding of human oncogenesis and the development of new therapeutic and biomarker targets with clinical potential.
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    The rational development of molecularly imprinted polymer-based sensors for protein detection.
    (Royal Society of Chemistry, 2011-02-22T00:00:00Z) Whitcombe, Michael J.; Chianella, Iva; Larcombe, Lee; Piletsky, Sergey A.; Noble, James; Porter, Robert; Horgan, Adrian
    The detection of specific proteins as biomarkers of disease, health status, environmental monitoring, food quality, control of fermenters and civil defence purposes means that biosensors for these targets will become increasingly more important. Among the technologies used for building specific recognition properties, molecularly imprinted polymers (MIPs) are attracting much attention. In this critical review we describe many methods used for imprinting recognition for protein targets in polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors (277 references).