MutS as a tool for mutation detection.

Stanisławska-Sachadyn, Anna; Sachadyn, Paweł

doi:10.18388/abp.2005_3417

Cited by 17 publications

(10 citation statements)

References 51 publications

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“…In addition, the nucleic acid enzymes have been applied for detecting SNPs. Coupled with DNA probes, MutS protein (DNA mismatch binding protein), − nucleases, , polymerases, , and ligases have been successfully used for SNP identification with high efficiency. Especially, the ligation reaction is superior to the two formers with respect to specificity as the high discrimination ability of ligases to base-mismatched sequences. − Although these approaches have made great contributions for SNP identification, limitations, such as insufficient discrimination capability for SNP, time-consuming, and relatively high cost, still exist.…”

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confidence: 99%

Combination of DNA Ligase Reaction and Gold Nanoparticle-Quenched Fluorescent Oligonucleotides: A Simple and Efficient Approach for Fluorescent Assaying of Single-Nucleotide Polymorphisms

Wang

et al. 2010

Anal. Chem.

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A new fluorescent sensing approach for detection of single-nucleotide polymorphisms (SNPs) is proposed based on the ligase reaction and gold nanoparticle (AuNPs)-quenched fluorescent oligonucleotides. The design exploits the strong fluorescence quenching of AuNPs for organic dyes and the difference in noncovalent interactions of the nanoparticles with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), where ssDNA can be adsorbed onto the surface of AuNPs while dsDNA cannot be. In the assay, two half primer DNA probes, one being labeled with a dye and the other being phosphorylated, were first incubated with a target DNA template. In the presence of DNA ligase, the two captured ssDNAs are linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of AuNPs, the fluorescence of dye-tagged DNA probe will be efficiently quenched unless the perfectly matched DNA target is present. To demonstrate the feasibility of this design, the performance of SNP detection using two different DNA ligases, T4 DNA ligase and Escherichia coli DNA ligase, were investigated. In the case of T4 DNA ligase, the signal enhancement of the dye-tagged DNA for perfectly matched DNA target is 4.6-fold higher than that for the single-base mismatched DNA. While in the presence of E. coli DNA ligase, the value raises to be 30.2, suggesting excellent capability for SNP discrimination.

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confidence: 99%

Combination of DNA Ligase Reaction and Gold Nanoparticle-Quenched Fluorescent Oligonucleotides: A Simple and Efficient Approach for Fluorescent Assaying of Single-Nucleotide Polymorphisms

Wang

et al. 2010

Anal. Chem.

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“…The method, proposed here, was used first in order to circumvent the problem of PCR contaminations occurring in the course of investigations on MutS protein (Sachadyn et al 2000 ; Stanisławska-Sachadyn et al 2005 ; Stanisławska-Sachadyn and Sachadyn 2005 ; Stanisławska-Sachadyn et al 2006 ; Stanisławska-Sachadyn et al 2003 ) where 69 bp amplicons were quantitated with real-time PCR in order to estimate the amounts of DNA bound by the protein. As mentioned above, all efforts, such as applying all good laboratory practices, replacement of PCR reagents and materials, and trying an alternative PCR master-mix, were ineffective to eradicate the persisting contaminations.…”

Section: Resultsmentioning

confidence: 99%

A simple modification of PCR thermal profile applied to evade persisting contamination

Banasik

Stanisławska-Sachadyn

Sachadyn

2016

J Appl Genetics

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The polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem of persisting PCR contamination is reported. The principle of this approach lies in shortening the steps of denaturation, annealing, and elongation in the PCR thermal cycle. The modification leads to the radical decline of false positive signals obtained for the no-template controls without affecting the detection of target PCR products. In the model experiments presented here, the signal of negative control was shifted by about ten cycles up above those for the examined samples so that it could be neglected. We do not recommend this solution in PCR diagnostics, where the sensitivity of detection is of the highest priority. However, the approach could be useful to pass by the problem of persisting contamination in quantitative PCR, where the range of quantitation is usually much above the limits of detection.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-015-0336-z) contains supplementary material, which is available to authorized users.

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“…MutS recognizes a mispaired base in DNA, which triggers a sequence of events resulting in mismatch correction. A number of methods employing MutS for DNA mismatch detection have been developed [ 8 – 11 ]. Here we present a microplate assay that could be a convenient solution for the preliminary detection of DNA mismatch binding activity.…”

Section: Introductionmentioning

confidence: 99%

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein

Banasik

Sachadyn

2016

Mol Biotechnol

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A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5–10 pmol) and DNA (0.1–10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA–protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

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MutS as a tool for mutation detection.

Cited by 17 publications

References 51 publications

Combination of DNA Ligase Reaction and Gold Nanoparticle-Quenched Fluorescent Oligonucleotides: A Simple and Efficient Approach for Fluorescent Assaying of Single-Nucleotide Polymorphisms

Combination of DNA Ligase Reaction and Gold Nanoparticle-Quenched Fluorescent Oligonucleotides: A Simple and Efficient Approach for Fluorescent Assaying of Single-Nucleotide Polymorphisms

A simple modification of PCR thermal profile applied to evade persisting contamination

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein

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