Base excision-initiated terminal deoxynucleotide transferase-assisted amplification for simultaneous detection of multiple DNA glycosylases.

Tytuł:: Base excision-initiated terminal deoxynucleotide transferase-assisted amplification for simultaneous detection of multiple DNA glycosylases.
Autorzy:: Sun Y; School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China.
Zang L; School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China.
Lu J; School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China. .
Źródło:: Analytical and bioanalytical chemistry [Anal Bioanal Chem] 2022 May; Vol. 414 (11), pp. 3319-3327. Date of Electronic Publication: 2022 Mar 12.
Typ publikacji:: Journal Article
Język:: English
Imprint Name(s):: Original Publication: Heidelberg : Springer-Verlag, 2002-
MeSH Terms:: DNA Nucleotidylexotransferase*
Uracil-DNA Glycosidase*/analysis
DNA Repair ; DNA-Directed DNA Polymerase ; HeLa Cells ; Humans
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Grant Information:: 21675030 National Natural Science Foundation of China
Contributed Indexing:: Keywords: DNA glycosylase; Multiplex detection; Signal amplification; Terminal deoxynucleotidyl transferase
Substance Nomenclature:: EC 2.7.7.31 (DNA Nucleotidylexotransferase)
EC 2.7.7.7 (DNA-Directed DNA Polymerase)
EC 3.2.2.- (Uracil-DNA Glycosidase)
Entry Date(s):: Date Created: 20220312 Date Completed: 20220421 Latest Revision: 20220716
Update Code:: 20240105
DOI:: 10.1007/s00216-022-03978-9
PMID:: 35277739
: Czasopismo naukowe

Pełny tekst

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10 -3 U/ml. Moreover, it could be applied for analyzing DNA glycosylase activity in complex HeLa cell lysate samples. Therefore, this strategy possesses the advantages of high sensitivity, specificity, and multiplex, holding great potential for DNA glycosylase-related biomedical research.
(© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

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