Issue 3, 2005

Using molecular beacon to monitor activity of E. coli DNA ligase

Abstract

NAD+-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD+-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 × 10−4 Weiss Unit mL−1 to 0.4 Weiss Unit mL−1 and the detection limit of 4.0 × 10−4 Weiss Unit mL−1.

Graphical abstract: Using molecular beacon to monitor activity of E. coli DNA ligase

Article information

Article type
Paper
Submitted
10 Sep 2004
Accepted
30 Nov 2004
First published
13 Jan 2005

Analyst, 2005,130, 350-357

Using molecular beacon to monitor activity of E. coli DNA ligase

L. Liu, Z. Tang, K. Wang, W. Tan, J. Li, Q. Guo, X. Meng and C. Ma, Analyst, 2005, 130, 350 DOI: 10.1039/B413959C

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements