Label-free voltammetric detection of single nucleotide
mismatches recognized by MutS protein

J 2007

Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein

MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK, Miroslav FOJTA et. al.

Základní údaje

Originální název

Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein

Název česky

Voltametrická detekce změn jednoho nukletidu pomoci MutS proteinu bez pouziti znacek

Autoři

MASAŘÍK, Michal (203 Česká republika), K. CAHOVÁ (203 Česká republika), René KIZEK (203 Česká republika, garant), Emil PALEČEK (203 Česká republika) a Miroslav FOJTA (203 Česká republika)

Vydání

ANALYTICAL AND BIOANALYTICAL CHEMISTRY, HEIDELBERG, GERMANY, SPRINGER-VERLAG HEIDELBERG, 2007, 1618-2642

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

Genetika a molekulární biologie

Stát vydavatele

Německo

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 2.867

Kód RIV

RIV/00216224:14110/07:00022108

Organizační jednotka

Lékařská fakulta

UT WoS

000245292200032

Klíčová slova anglicky

voltammetric detection;single nucleotide mismatches; MutS protein

Štítky

MutS protein, single nucleotide mismatches, voltammetric detection

Příznaky

Recenzováno

Změněno: 25. 6. 2009 10:45, prof. RNDr. Michal Masařík, Ph.D.

Anotace

ORIG CZ

V originále

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.

Česky

Voltametrická detekce změn jednoho nukletidu pomoci MutS proteinu bez pouziti znacek

Návaznosti

LC06035, projekt VaV

Název: Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy. Nové nástroje pro genomiku, proteomiku a biomedicínu.

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy. Nové nástroje pro genomiku, proteomiku a biomedicínu

Citovat

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.

@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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