Biomedical subjects

Anthony J Brookes

Publications and source records attributed to Anthony J Brookes.

43 records · Page 3Linked to original sources

PubMed2003

Scoring insertion-deletion polymorphisms by dynamic allele-specific hybridization.

Genome variation provides researchers with thousands of markers with which to study human demographic history and phenotypes. Insertion-deletion (indel) polymorphism is an important and abundant form of human genome variation, and convenient methods for genotyping indels are therefore needed. Here we evaluate dynamic allele-specific hybridization (DASH) for its ability to score indels. Evaluation of six model indel DASH assays based on synthetic oligonucleotides showed that length differences of 1-5 bp were accurately scored. Only single probes were required to assay indels of 3-4 bp or less, while longer indels tended to require the use of both allele probes serially. The best results were obtained by central placing of the probe over the indel. Model study findings were confirmed by running indel DASH assays upon PCR-amplified targets representing four polymorphisms from Alzheimer's disease candidate genes APBB1 and LRP1. These indels were genotyped in a set of 121 patients and 156 controls. While no disease association was found, the data quality confirmed that DASH is a robust and useful procedure for genotyping indels of the size range typically found in the human genome.

Alleles↗

PubMed2002

A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans.

Systemic lupus erythematosus (SLE, OMIM 152700) is a complex autoimmune disease that affects 0.05% of the Western population, predominantly women. A number of susceptibility loci for SLE have been suggested in different populations, but the nature of the susceptibility genes and mutations is yet to be identified. We previously reported a susceptibility locus (SLEB2) for Nordic multi-case families. Within this locus, the programmed cell death 1 gene (PDCD1, also called PD-1) was considered the strongest candidate for association with the disease. Here, we analyzed 2,510 individuals, including members of five independent sets of families as well as unrelated individuals affected with SLE, for single-nucleotide polymorphisms (SNPs) that we identified in PDCD1. We show that one intronic SNP in PDCD1 is associated with development of SLE in Europeans (found in 12% of affected individuals versus 5% of controls; P = 0.00001, r.r. (relative risk) = 2.6) and Mexicans (found in 7% of affected individuals versus 2% of controls; P = 0.0009, r.r. = 3.5). The associated allele of this SNP alters a binding site for the runt-related transcription factor 1 (RUNX1, also called AML1) located in an intronic enhancer, suggesting a mechanism through which it can contribute to the development of SLE in humans.

3' Untranslated Regions↗

PubMed2002

Evaluation of multiple presenilin 2 SNPs for association with early-onset sporadic Alzheimer disease.

The presenilin genes encode proteins that modify, mediate, or perform similar functions to gamma-secretase, the enzyme responsible for converting amyloid beta precursor protein (APP) into beta-amyloid. Mutations in the presenilin genes cause an increased production of Abeta42, the aberrant form of beta-amyloid found in the neural plaques of Alzheimer disease patients. Previously reported association studies of presenilin 2 (PSEN2) polymorphisms with early-onset Alzheimer disease (EOAD) have produced contradictory results. In an effort to resolve these differences, we tested eight single nucleotide polymorphisms in and around the 3' region of the PSEN2 gene for association with EOAD. An initial set of Scottish EOAD cases (n = 121) and controls (n = 152) was screened using the genotyping method dynamic allele-specific hybridization (DASH). No significant differences were seen between allele or genotype frequencies of cases and controls. However, when conditioned on the risk allele (epsilon 4) APOE, three polymorphisms showed allelic association with a P value below 0.05. These same polymorphisms were in near 100% linkage disequilibrium with each other (P < 5 x 10(-5)), and in each, one of the homozygous genotypes was absent in controls but present in the cases. Replication in an independent set of Scottish EOAD cases (n = 84) and controls (n = 173) did not confirm this finding. From this study we find no evidence to suggest that variations in the PSEN2 gene pose as major risk factors for sporadic EOAD.

3' Untranslated Regions↗

PubMed2002

Effect of oligonucleotide truncation on single-nucleotide distinction by solid-phase hybridization.

Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.

Animals↗

PubMed2002

4th International Meeting on Single Nucleotide Polymorphism and Complex Genome Analysis. Various uses for DNA variations.

At the 4th International Meeting on Single Nucleotide Polymorphism and Complex Genome Analysis (Stockholm, Sweden, 10th-14th October 2001), approximately 100 scientists from more than 20 nations undertook a probing review of latest developments in the field. Despite impressive and still ongoing activities towards SNP discovery and validation, plus efforts towards haplotype exploitation, it was clear that supporting technologies for genotyping are way behind where they need to be. Innate complexity and large variances in aspects of genome function together pose immense challenges that are difficult to surmount in the human situation. In contrast, studies in simpler organisms and population/evolutionary genetics studies are yielding important new insights. Breakthroughs that are being made in understanding the genetic etiology of complex disease tend to involve genes of larger effect or extremely well merited candidates. Linkage studies and proximal phenotypes are being recommended, though the best way forward is still hotly debated. Consequently, many diverse and ambitious projects are underway, from which the data itself will eventually show what is and is not possible.

Genome, Human↗

PubMed2002

iFRET: an improved fluorescence system for DNA-melting analysis.

Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.

Alleles↗

PubMed2002

Creating arrays by centrifugation.

We describe afast, low-cost, and reliable way of creating arrays from sample molecules of interest present within microformatted sample vessels (such as 1536-well microplates). The principle involves simple centrifugal transfer of molecules of interest onto a solid planar or membrane surfaces placed over the initial sample vessel. Tools and procedures are presented that validate the robustness and precision of this facile solution to an otherwise difficult problem in modern molecular genetics. The availability of transferred DNA molecules for hybridization is also demonstrated. In conclusion, this "centrifugal-array" concept should help research studies to be applied on ever-greater scales with very simple machinery.

Centrifugation↗