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; K. CAHOVÁ; René KIZEK; Emil PALEČEK a Miroslav FOJTA

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

Označené pro přenos do RIV

Ano

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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