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MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK and Miroslav FOJTA. Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. HEIDELBERG, GERMANY: SPRINGER-VERLAG HEIDELBERG, 2007, vol. 388, No 1, p. 259-270. ISSN 1618-2642.
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Basic information
Original name Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein
Name in Czech Voltametrická detekce změn jednoho nukletidu pomoci MutS proteinu bez pouziti znacek
Authors MASAŘÍK, Michal (203 Czech Republic), K. CAHOVÁ (203 Czech Republic), René KIZEK (203 Czech Republic, guarantor), Emil PALEČEK (203 Czech Republic) and Miroslav FOJTA (203 Czech Republic).
Edition ANALYTICAL AND BIOANALYTICAL CHEMISTRY, HEIDELBERG, GERMANY, SPRINGER-VERLAG HEIDELBERG, 2007, 1618-2642.
Other information
Original language English
Type of outcome Article in a journal
Field of Study Genetics and molecular biology
Country of publisher Germany
Confidentiality degree is not subject to a state or trade secret
Impact factor Impact factor: 2.867
RIV identification code RIV/00216224:14110/07:00022108
Organization unit Faculty of Medicine
UT WoS 000245292200032
Keywords in English voltammetric detection;single nucleotide mismatches; MutS protein
Tags MutS protein, single nucleotide mismatches, voltammetric detection
Tags Reviewed
Changed by Changed by: prof. RNDr. Michal Masařík, Ph.D., učo 21142. Changed: 25/6/2009 10:45.
Abstract
MutS, a protein involved in DNA mismatch repair, recognizes mispaired and unpaired bases in duplex DNA. We have previously used MutS in an electrochemical double-surface technique (DST) for in-vitro detection of point mutations in DNA. The DST involved binding of unlabeled MutS to DNA heteroduplexes at the surface of magnetic beads followed by a highly sensitive electrochemical determination of the protein by measurement of a catalytic protein signal (peak H) at mercury electrodes. Detection of MutS using a peak resulting from oxidation of tyrosine and tryptophan residues of the protein at a carbon-paste electrode (CPE) was also possible but was approximately three orders of magnitude less sensitive. In this work we present an optimized technique for ex-situ voltammetric determination of MutS at a CPE. Choice of optimum experimental conditions (pH of supporting electrolyte, square-wave voltammetry settings, etc.) resulted in substantial improvement of the sensitivity of the assay, enabling detection of approximately 140 pg (1.6 fmol protein monomer) MutS in a 5-mu L sample. The sensitivity was increased further by acid hydrolysis of the protein before measurement. The hydrolyzed protein was detectable down to 5 pg (approx. 56 amol) MutS in 5 mu L solution. By using the DST combined with determination of the bound unlabeled MutS at the CPE we demonstrated selective interactions of the protein with single-base mismatches and discrimination among different base mispairs in 30-mer or 95-mer DNA duplexes. In agreement with previous studies, binding of the protein to the 30-mer substrates followed the trend G:T >> C:A > A:A > C:T > homoduplex. The electrochemical data were confirmed by use of an independent technique-a quartz-crystal microbalance for real-time monitoring of MutS interactions with DNA duplexes containing different base mispairs. By using the electrochemical DST a G:T mismatch was detectable in up to 1000-fold excess of homoduplex DNA.
Abstract (in Czech)
Voltametrická detekce změn jednoho nukletidu pomoci MutS proteinu bez pouziti znacek
Links
LC06035, research and development projectName: Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy. Nové nástroje pro genomiku, proteomiku a biomedicínu.
Investor: Ministry of Education, Youth and Sports of the CR, Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis. New Tools for Genomics, Proteomics and Biomedicine
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