A Sequencing Method Based on Real-Time Pyrophosphate

Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål

doi:10.1126/science.281.5375.363

Cited by 1,329 publications

(805 citation statements)

References 14 publications

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“…The cycling conditions used were an initial 96°C for 15 minutes to activate the hot start Taq, followed by 35 cycles of 30 s denaturation at 96°C, 1 min 30 s annealing at 53°C and 1 min 30 s extension at 72°C. Amplification products were SNP genotyped by pyrosequencing [26]. Genbank accession numbers of sequences containing all SNPs and location of SNPs are available [18,22].…”

Section: Methodsmentioning

confidence: 99%

Compilation of a panel of informative single nucleotide polymorphisms for bovine identification in the Northern Irish cattle population

et al. 2010

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Background Animal identification is pivotal in governmental agricultural policy, enabling the management of subsidy payments, movement of livestock, test scheduling and control of disease. Advances in bovine genomics have made it possible to utilise inherent genetic variability to uniquely identify individual animals by DNA profiling, much as has been achieved with humans over the past 20 years. A DNA profiling test based on bi-allelic single nucleotide polymorphism (SNP) markers would offer considerable advantages over current short tandem repeat (STR) based industry standard tests, in that it would be easier to analyse and interpret. In this study, a panel of 51 genome-wide SNPs were genotyped across panels of semen DNA from 6 common breeds for the purposes of ascertaining allelic frequency. For SNPs on the same chromosome, the extent of linkage disequilbrium was determined from genotype data by Expectation Maximization (EM) algorithm. Minimum probabilities of unique identification were determined for each breed panel. The usefulness of this SNP panel was ascertained by comparison to the current bovine STR Stockmarks II assay. A statistically representative random sampling of bovine animals from across Northern Ireland was assembled for the purposes of determining the population allele frequency for these STR loci and subsequently, the minimal probability of unique identification they conferred in sampled bovine animals from Northern Ireland. Results 6 SNPs exhibiting a minor allele frequency of less than 0.2 in more than 3 of the breed panels were excluded. 2 Further SNPs were found to reside in coding areas of the cattle genome and were excluded from the final panel. The remaining 43 SNPs exhibited genotype frequencies which were in Hardy Weinberg Equilibrium. SNPs on the same chromosome were observed to have no significant linkage disequilibrium/allelic association. Minimal probabilities of uniquely identifying individual animals from each of the breeds were obtained and were observed to be superior to those conferred by the industry standard STR assay. Conclusions The 43 SNPs characterised herein may constitute a starting point for the development of a SNP based DNA identification test for European cattle.

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Section: Methodsmentioning

confidence: 99%

Compilation of a panel of informative single nucleotide polymorphisms for bovine identification in the Northern Irish cattle population

et al. 2010

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“…(4) Recently, two modified bisulfite-treated DNA methylation-mapping techniques have been developed; they are "pyrosequencing" [76,77] and "RNase T1 cleavage /matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)" [78]. Pyrosequencing is a real-time sequencing technology, which detects the release of pyrophosphate (PPi) during nucleotide incorporation, the quantitative conversion of pyrophosphate to ATP by sulfurylase, and the subsequent production of visible light by luciferase [79]. This method can measure as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides.…”

Section: Pcr-based Single Gene Dna Methylation Detectionmentioning

confidence: 99%

Epigenetic changes in virus-associated human cancers

Leu

Chang

2005

Cell Res

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Epigenetics of human cancer becomes an area of emerging research direction due to a growing understanding of specific epigenetic pathways and rapid development of detection technologies. Aberrant promoter hypermethylation is a prevalent phenonmena in human cancers. Tumor suppressor genes are often hypermethylated due to the increased activity or deregulation of DNMTs. Increasing evidence also reveals that viral genes are one of the key players in regulating DNA methylation. In this review, we will focus on hypermethylation and tumor suppressor gene silencing and the signal pathways that are involved, particularly in cancers closely associated with the hepatitis B virus, simian virus 40 (SV40), and Epstein-Barr virus. In addition, we will discuss current technologies for genome-wide detection of epigenetically regulated targets, which allow for systematic DNA hypermethylation analysis. The study of epigenetic changes should provide a global view of gene profile in cancer, and epigenetic markers could be used for early detection, prognosis, and therapy of cancer.

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“…[7][8][9] In MS, association studies have investigated both positional candidate genes and functional candidate genes, including genes encoding immunoglobulins and T-cell receptors, HLA molecules and myelin antigens, and chemokines, interleukins and other cytokines; however, to date, with the exception of various HLA class II haplotypes, no candidate gene has reproducibly been shown to be associated with MS. 10 In this study, we have genotyped 123 SNPs, in a total of 66 candidate genes, in up to 672 MS patients and 672 controls, using the Pyrosequencing technique 11 and a two-stage study design. In the first stage, we made use of a limited number of markers and a limited number of patients and controls; in selecting polymorphisms in this stage, we sought to choose exonic SNPs, giving highest priority to SNPs encoding nonsynonymous nucleotide changes.…”

Section: Introductionmentioning

confidence: 99%

Two genes encoding immune-regulatory molecules (LAG3 and IL7R) confer susceptibility to multiple sclerosis

et al. 2005

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Multiple sclerosis (MS) is a T-cell-mediated disease of the central nervous system, characterized by damage to myelin and axons, resulting in progressive neurological disability. Genes may influence susceptibility to MS, but results of association studies are inconsistent, aside from the identification of HLA class II haplotypes. Whole-genome linkage screens in MS have both confirmed the importance of the HLA region and uncovered non-HLA loci that may harbor susceptibility genes. In this twostage analysis, we determined genotypes, in up to 672 MS patients and 672 controls, for 123 single-nucleotide polymorphisms (SNPs) in 66 genes. Genes were chosen based on their chromosomal positions or biological functions. In stage one, 22 genes contained at least one SNP for which the carriage rate for one allele differed significantly (Po0.08) between patients and controls. After additional genotyping in stage two, two genes-each containing at least three significantly (Po0.05) associated SNPs-conferred susceptibility to MS: LAG3 on chromosome 12p13, and IL7R on 5p13. LAG3 inhibits activated T cells, while IL7R is necessary for the maturation of T and B cells. These results imply that germline allelic variation in genes involved in immune homeostasis-and, by extension, derangement of immune homeostasis-influence the risk of MS.

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A Sequencing Method Based on Real-Time Pyrophosphate

Cited by 1,329 publications

References 14 publications

Compilation of a panel of informative single nucleotide polymorphisms for bovine identification in the Northern Irish cattle population

Compilation of a panel of informative single nucleotide polymorphisms for bovine identification in the Northern Irish cattle population

Epigenetic changes in virus-associated human cancers

Two genes encoding immune-regulatory molecules (LAG3 and IL7R) confer susceptibility to multiple sclerosis

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