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@article{1187347,
author = {Stejskalová, Eva and Horáková, Petra and Vacek, Jan and Bowater, Richard and Fojta, Miroslav},
article_location = {Heidelberg},
article_number = {17},
doi = {http://dx.doi.org/10.1007/s00216-014-7811-y},
keywords = {Electrochemistry; Enzyme labeling; DNA ligase; Nicked DNA; Magnetic beads; Immobilized assays},
language = {eng},
issn = {1618-2642},
journal = {Analytical and Bioanalytical Chemistry},
title = {Enzyme-linked electrochemical DNA ligation assay using magnetic beads},
url = {http://link.springer.com/article/10.1007%2Fs00216-014-7811-y},
volume = {406},
year = {2014}
}
TY - JOUR
ID - 1187347
AU - Stejskalová, Eva - Horáková, Petra - Vacek, Jan - Bowater, Richard - Fojta, Miroslav
PY - 2014
TI - Enzyme-linked electrochemical DNA ligation assay using magnetic beads
JF - Analytical and Bioanalytical Chemistry
VL - 406
IS - 17
SP - 4129-4136
EP - 4129-4136
PB - Springer
SN - 16182642
KW - Electrochemistry
KW - Enzyme labeling
KW - DNA ligase
KW - Nicked DNA
KW - Magnetic beads
KW - Immobilized assays
UR - http://link.springer.com/article/10.1007%2Fs00216-014-7811-y
L2 - http://link.springer.com/article/10.1007%2Fs00216-014-7811-y
N2 - DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5 biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3 digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.
ER -
STEJSKALOVÁ, Eva, Petra HORÁKOVÁ, Jan VACEK, Richard BOWATER a Miroslav FOJTA. Enzyme-linked electrochemical DNA ligation assay using magnetic beads. \textit{Analytical and Bioanalytical Chemistry}. Heidelberg: Springer, 2014, roč.~406, č.~17, s.~4129-4136. ISSN~1618-2642. Dostupné z: https://dx.doi.org/10.1007/s00216-014-7811-y.
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