期刊
METHODS
卷 106, 期 -, 页码 66-75出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2016.03.018
关键词
DNAzyme; In vitro selection; RNA cleavage; Bacterial detection; Biosensor; Clostridium difficile
资金
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Canadian Institutes of Health Research (CIHR)
DNAzymes refer to single-stranded DNA molecules with catalytic activity and can be isolated from synthetic random-sequence DNA pools using the technique of in vitro selection. DNAzymes that cleave RNA, known as RNA-cleaving DNAzymes, represent one of the best-studied classes of DNAzymes and have been widely used for the development of biosensors and bioassays for various analytes. We have been interested in developing RNA-cleaving DNAzymes as bacterial sensors and these DNAzymes are engineered to perform three linked functions: recognition of a bacterial biomarker, RNA cleavage, and fluorescence generation. These fluorogenic DNAzymes emit fluorescence automatically in the presence of a bacterium of interest and can be used to set up a simple mix-and-read assay to detect this bacterium. In this article, we will discuss this DNAzyme system and present a proven strategy for isolating highly specific bacteria -responding DNAzyme probes from random-sequence DNA pools. We will also provide an in vitro selection protocol successfully used to derive RNA-cleaving fluorogenic DNAzyme probes that are capable of recognizing a targeted strain of Clostridium difficile. (C) 2016 Elsevier Inc. All rights reserved.
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