STEJSKALOVÁ, Eva, Petra HORÁKOVÁ, Jan VACEK, Richard BOWATER and Miroslav FOJTA. Enzyme-linked electrochemical DNA ligation assay using magnetic beads. Analytical and Bioanalytical Chemistry. Heidelberg: Springer, vol. 406, No 17, p. 4129-4136. ISSN 1618-2642. doi:10.1007/s00216-014-7811-y. 2014.
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Basic information
Original name Enzyme-linked electrochemical DNA ligation assay using magnetic beads
Authors STEJSKALOVÁ, Eva (203 Czech Republic), Petra HORÁKOVÁ (203 Czech Republic), Jan VACEK (203 Czech Republic), Richard BOWATER (826 United Kingdom of Great Britain and Northern Ireland) and Miroslav FOJTA (203 Czech Republic, guarantor, belonging to the institution).
Edition Analytical and Bioanalytical Chemistry, Heidelberg, Springer, 2014, 1618-2642.
Other information
Original language English
Type of outcome Article in a journal
Field of Study 10406 Analytical chemistry
Country of publisher Germany
Confidentiality degree is not subject to a state or trade secret
WWW URL
Impact factor Impact factor: 3.436
RIV identification code RIV/00216224:14740/14:00075856
Organization unit Central European Institute of Technology
Doi http://dx.doi.org/10.1007/s00216-014-7811-y
UT WoS 000337787400010
Keywords in English Electrochemistry; Enzyme labeling; DNA ligase; Nicked DNA; Magnetic beads; Immobilized assays
Tags kontrola MP, rivok
Tags International impact, Reviewed
Changed by Changed by: Martina Prášilová, učo 342282. Changed: 11/3/2015 08:32.
Abstract
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.
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