Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

Stanisławska-Sachadyn, Anna; Paszko, Z; Kluska, Anna; Skasko, E.; Sromek, Maria; Bałabas, Aneta; Janiec–Jankowska, Aneta; Wiśniewska, A; Kur, Józef; Sachadyn, Paweł

doi:10.1016/j.mrfmmm.2004.10.001

Cited by 9 publications

(11 citation statements)

References 30 publications

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“…If heterozygotic alleles are examined (i.e. one allele contains a mutation, the other does not), the addition of the reference DNA is not necessary (Stanisławska-Sachadyn et al, 2005).…”

Section: Muts Based Methods For Mutation Detectionmentioning

confidence: 99%

“…Formation of a MutS-DNA complex may be detected in many ways: direct microscopic observation of complexes using atomic force microscopy, AFM (Sun & Yokota, 2000), the DNA mobility shi� in electrophoresis (Lishanski et al, 1994;Takamatsu et al, 1996;Stanisławska-Sachadyn et al, 2005), DNA protection by MutS against nuclease digestion (Ellis et al, 1994;Sachadyn et al, 2000), the detection of MutS-DNA interactions on a solid phase, including filter assays with radioactive (Whitehouse et al, 1997) or biotin (Wagner, et al, 1995) labelled DNA, and on chip detection (Gotoh et al, 1997;Behrensdorf et al, 2000;Bi et al, 2003). The detection of MutS was enabled by using fluorescent MutS (cyanine labelled) or one fused with reporter domains like a biotinylated tag (Geschwind et al, 1996) or GFP (Stanisławska-Sachadyn et al, J Biotechnol, accepted).…”

Section: Muts Based Methods For Mutation Detectionmentioning

confidence: 99%

“…In vitro, MutS recognises mispaired bases and unpaired bases, small DNA insertion/deletion loops of one to three (Whitehouse et al, 1997), four (Parker & Marinus, 1992) or five nucleotides (Stanisławska-Sachadyn et al, 2005). MutS also binds damaged DNA, like DNA adducts (Ducke� et al, 1996;Johnson et al, 1999).…”

Section: Mutationsmentioning

confidence: 99%

“…We used PCR fragments in the range of 200-700 bp. In our assay (Stanisławska- Sachadyn et al, 2005), the PCR products were neither labelled nor purified prior to examination. The MutS-DNA complexes were electrophoresed in polyacrylamide gels stained with a sensitive fluorescent dye, SYBR-Gold (Fig.…”

Section: Dna Retardationmentioning

confidence: 99%

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MutS as a tool for mutation detection.

Stanisławska-Sachadyn

Sachadyn²

2005

Acta Biochim Pol

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MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 microg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 microg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like beta-galactosidase or GFP. Very low detection limits for beta-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.

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“…If heterozygotic alleles are examined (i.e. one allele contains a mutation, the other does not), the addition of the reference DNA is not necessary (Stanisławska-Sachadyn et al, 2005).…”

Section: Muts Based Methods For Mutation Detectionmentioning

confidence: 99%

Section: Muts Based Methods For Mutation Detectionmentioning

confidence: 99%

Section: Mutationsmentioning

confidence: 99%

Section: Dna Retardationmentioning

confidence: 99%

See 2 more Smart Citations

MutS as a tool for mutation detection.

Stanisławska-Sachadyn

Sachadyn²

2005

Acta Biochim Pol

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“…A number of methods addressing the cardinal problem of detecting DNA–protein complexes have been developed. Gel retardation [1, 2] or nitrocellulose filter binding assays [3] are the examples of simple, but rather time-demanding, approaches. The advanced methods such as surface plasmon resonance [4], laser scattering or atomic force microscopy [5] open more possibilities but they depend on sophisticated instruments.…”

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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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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Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

Cited by 9 publications

References 30 publications

MutS as a tool for mutation detection.

MutS as a tool for mutation detection.

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

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

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