| MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK a Miroslav FOJTA. Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. HEIDELBERG, GERMANY: SPRINGER-VERLAG HEIDELBERG, 2007, roč. 388, č. 1, s. 259-270. ISSN 1618-2642. |
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@article{718630,
author = {Masařík, Michal and Cahová, K. and Kizek, René and Paleček, Emil and Fojta, Miroslav},
article_location = {HEIDELBERG, GERMANY},
article_number = {1},
keywords = {voltammetric detection;single nucleotide mismatches; MutS protein},
language = {eng},
issn = {1618-2642},
journal = {ANALYTICAL AND BIOANALYTICAL CHEMISTRY},
title = {Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein},
volume = {388},
year = {2007}
}
TY - JOUR
ID - 718630
AU - Masařík, Michal - Cahová, K. - Kizek, René - Paleček, Emil - Fojta, Miroslav
PY - 2007
TI - Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein
JF - ANALYTICAL AND BIOANALYTICAL CHEMISTRY
VL - 388
IS - 1
SP - 259-270
EP - 259-270
PB - SPRINGER-VERLAG HEIDELBERG
SN - 16182642
KW - voltammetric detection;single nucleotide mismatches
KW - MutS protein
N2 - 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.
ER -
MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK a Miroslav FOJTA. Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein. \textit{ANALYTICAL AND BIOANALYTICAL CHEMISTRY}. HEIDELBERG, GERMANY: SPRINGER-VERLAG HEIDELBERG, 2007, roč.~388, č.~1, s.~259-270. ISSN~1618-2642.
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