Proofreading genotyping assays and electrochemical detection of SNPs

King, Garry C.; Giusto, Daniel A. Di; Wlassoff, Wjatschesslaw A.; Giesebrecht, Susanne; Flening, Eleanor; Tyrelle, Gregory D.

doi:10.1002/humu.20034

Cited by 12 publications

(12 citation statements)

References 19 publications

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“…the product obtained from the cleaved probes can be extended with fluorescence labeled dNTP or ddNTP. In the future, we intend to improve our method by replacing allele specific extension with single base extension which can be carried out with fluorescence labeled ddNTPs (3,9). Thus, the assay could be multiplexed by varying the length of the probes which are used in single base extension.…”

Section: Prospects Of the Rcae Methodsmentioning

confidence: 99%

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

Hou

Liu

2012

Biotechnology & Biotechnological Equipment

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Section: Prospects Of the Rcae Methodsmentioning

confidence: 99%

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

Hou

Liu

2012

Biotechnology & Biotechnological Equipment

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“…The aforementioned data illustrates the powerful mutation detection ability of the novel on/off switch, indicating its potential in the analysis of both SNP and other types of mutations. Except for the deletion or insertion of identical short repeats, the application of this on/off switch provides an efficient and high throughput compatible assay for mutation analysis [4][5][6][7][8][9][10][11]16] . Detection of rare alleles using the novel on/off switch The novel SNP-operated on/off switch is more sensitive than many conventional mutation analysis methods, including preferential amplification of the mutant allele, preferential destruction of the wild-type allele and spatial separation of mutant from wild-type alleles, by virtue of its use of Exo + high-fidelity DNA polymerases.…”

Section: Genomapping Applications Other Than Snp Assaymentioning

confidence: 99%

“…Based on the new model of this proofreading mechanism (Figure 1), polymerases with 3´ exonuclease function should have a higher base discrimination ability over exo-polymerases regardless of the properties of the substrates used. In addition to comparing nine different DNA polymerases, Di Giusto et al [6,7,14] evaluated the effect of dNTP(2´-deoxynucleotide-5´-triphosphate), ddNTP (2´,3´-dideoxynucleotide-5´-triphosphate), and acyNTP (acyclo-2´-deoxynucleotide-5´-triphosphate) on the accuracy of primer extension. The maintenance of high fidelity with ddNTP and acyNTP allows the Exo + polymerases to be applied in both exponential and linear primer extensions in SNP analysis.…”

Section: Introductionmentioning

confidence: 99%

Exo+ proofreading polymerases mediate genetic analysis and its application in biomedical studies1

Liao

Chen

Peng

et al. 2005

Acta Pharmacologica Sinica

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High-fidelity DNA polymerases mediate genotyping3´Terminal-labeled primer extension Terminal-labeled primer extension is a single nucleotide polymorphism (SNP) assay consisting of 3´ terminal-labeled, allele-specific primers and DNA polymerases with proofreading activity. Both 3´ terminal [ 3 H]-labeled and fluorescent-labeled primers have been successfully applied in genotyping analyses. The 3t erminal mismatched nucleotide that bears the signal to be detected was removed by the proofreading function, whereas the label was retained when the primer and template were perfectly matched. The terminal-labeled primer extension approach has several advantages over current SNP assays. The most significant advantage is that it greatly decreases false positive results by a direct consequence of the proofreading activity of Exo + polymerases. The second advantage of terminal-labeled primer extension is its high sensitivity.Terminally labeled primer extension harnesses the power of polymerase chain reaction (PCR) to improve the efficiency of genetic analysis [1][2][3][4][5][6][7] . SNP-triggered on/off switch Exo + polymerases together with 3´ phosphorothioate-modified mismatched primers work as an off switch in DNA polymerization. Phosphorothioate modification renders oligonucleotides nuclease-resistant, which blocks mismatch excision, a strategy widely used in antisense technology as well as in single base extension. For 3´ allele-specific primers with phosphorothioate modification, a perfectly matched primer turns on DNA polymerization, whereas mismatched primers turn it off, resulting in no product. This breakthrough observation of an on/off switch action has been repeatedly confirmed using either short artificial amplicons or natural genomic DNA templates. The off switch directly resulted from 3´exonu-clease activity and has been well supported by comparisons of a variety of DNA polymerases in both linear and expo- AbstractPolymerases with a proofreading function in their internal 3´ to 5´ exonuclease possess high fidelity for DNA replication both in vivo and in vitro. The obstacle facing Exo + polymerases for single nucleotide polymorphism (SNP) detection could be bypassed by using primer-3´-termini modification. This hypothesis has been well tested using three types of modified allele specific primers with: 3´ labeling, 3t o 5´exonuclease resistance, and 3´dehydroxylation. Accordingly, three new SNP assaying methods have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of Exo + polymerases. These new mutation detection assays are widely adaptable to a variety of platforms, including multi-well plate and microarray technologies. Application of Exo + polymerases to genetic analysis, including genotyping that is mostly relevant to pharmacogenetics, high-fidelity gene expression profiling, rare mutation detection and mutation load assay, will help to accelerate the pace of personalized medicine. In this review paper, we will first introduce three new assays that we have re...

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“…Garry King (The University of New South Wales, Sydney, Australia) presented a new process for manufacturing miniaturized devices, and their application to both electrophoretic size-separation and single-base extension assays. In this system, the extension products, combined with novel electrochemically tagged nucleotide substrates, or ''electrotides,'' can be directly detected on electrode surfaces [King et al, 2004]. Shigeori Takenaka (Kyushu University, Fukuoka, Japan) and Masahiko Amano (TUM Gene, Chiba, Japan) are developing a compact electrochemical array system.…”

Section: Microfluidic Devicesmentioning

confidence: 99%

Approaches for analyzing human mutations and nucleotide sequence variation: A report from the Seventh International Mutation Detection meeting, 2003

Syvänen

Taylor

2004

Hum. Mutat.

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The Seventh International Symposium on Mutations in the Human Genome, Mutation Detection 2003, was held during 2-6 July 2003 in Palm Cove near Cairns, Australia. The meeting was organized under the auspices of the Human Genome Organisation (HUGO) as a satellite meeting of the International World Congress of Genetics, held in Melbourne the following week. Meeting participants reported on advances in mutation detection technologies, including advances in high-throughput detection systems for SNP genotyping applicable to the international haplotype mapping project (HapMap); and bioinformatics tools, including databases for handling and processing growing amounts of genome variation data. This meeting report summarizes the presentations and cites related articles from the special issue of Human Mutation (Volume 23#5, May 2004; available online at www.wiley.com/humanmutation).

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Proofreading genotyping assays and electrochemical detection of SNPs

Cited by 12 publications

References 19 publications

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

Exo+ proofreading polymerases mediate genetic analysis and its application in biomedical studies1

Approaches for analyzing human mutations and nucleotide sequence variation: A report from the Seventh International Mutation Detection meeting, 2003

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