Direct and quantitative electrophoretic detection of covalently closed DNA circles formed by in vitro ligation

Akiko Mizutani; Masafumi Tanaka

doi:10.1016/j.ab.2018.05.026

Direct and quantitative electrophoretic detection of covalently closed DNA circles formed by in vitro ligation

Anal Biochem. 2018 Jul 15:553:54-56. doi: 10.1016/j.ab.2018.05.026. Epub 2018 May 29.

Authors

Akiko Mizutani¹, Masafumi Tanaka²

Affiliations

¹ Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan; Faculty of Health and Medical Science, Teikyo Heisei University, Tokyo, Japan.
² Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan. Electronic address: AKK18005@cc.u-tokai.ac.jp.

PMID: 29856979
DOI: 10.1016/j.ab.2018.05.026

Abstract

The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are difficult to analyze on conventional gels, they are often converted to nicked circles prior to electrophoresis. Herein, we present a sensitive and quantitative procedure for directly analyzing ligated closed circle DNA on agarose gels without additional treatments. Specifically, inclusion of GelStar dye in the gel allowed detection of ligated closed circle DNAs, which were likely super-twisted by being intercalated by GelStar, as discrete bands with good separation from linear DNA of the same sizes.

Keywords: Agarose gel electrophoresis; Closed relaxed circle; GelStar; Ligation; Prestain; Self-circularization.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Ligases / metabolism
DNA, Circular / chemistry*
Electrophoresis, Agar Gel / methods*
Fluorescence
Nucleic Acid Conformation

Substances

DNA, Circular
DNA Ligases