Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA.
Traditional methods to assay enzymatic cleavage of DNA are discontinuous and time consuming. In contrast, recently developed fluorescence methods are continuous and convenient. However, no fluorescence method has been developed for single-stranded DNA digestion. Here we introduce a novel method, based on molecular beacons, to assay single-stranded DNA cleavage by single strand-specific nucleases. A molecular beacon, a hairpin-shaped DNA probe labeled with a fluorophore and a quencher, is used as the substrate and enzymatic cleavage leads to fluorescence enhancement in the molecular beacon. This method permits real time detection of DNA cleavage and makes it easy to characterize the activity of DNA nucleases and to study the steady-state cleavage reaction kinetics. The excellent sensitivity, reproducibility and convenience will enable molecular beacons to be widely useful for the study of single-stranded DNA cleaving reactions.
Li
J J
JJ
Department of Chemistry and University of Florida Brain Institute, University of Florida, Gainesville, FL 32611, USA.
Geyer
R
R
Tan
W
W
D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D015342:DNA Probes; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases