Ethidium Bromide-Free DNA in Chimeric Gene Construction Involving Multiple DNA Segments

Short Communication

Austin J Plant Biol. 2015;1(1): 1002.

Ethidium Bromide-Free DNA in Chimeric Gene Construction Involving Multiple DNA Segments

Wun S Chao*

U.S. Department of Agricultural Research Service, Plant Science Research, Biosciences Research Laboratory, USA

*Corresponding author: Wun S Chao, U.S. Department of Agricultural Research Service, Plant Science Research, Biosciences Research Laboratory, Sunflower and Plant Biology Research Unit, 1605 Albrecht Blvd, Fargo, ND 58102-2765, USA

Received: September 16, 2014; Accepted: October 20, 2014; Published: October 27, 2014

Abstract

This manuscript describes a method of extracting DNA from agarose gels without exposure to ethidium bromide (EtBr) and UV radiation. Although it is well known that DNA exposed to EtBr and UV is less effective for cloning work, EtBr is still the most widely used staining agent today. Another newer and well-liked staining agent, SYBR Green, also requires UV radiation. Here I describe a simple method that uses these highly sensitive fluorescent agents to locate DNA for extraction, but does not expose the DNA itself to either fluorescent dyes or UV. Thus, the purified DNA can be used effectively for multi-segment ligation and regular cloning work.

Keywords: Ethidium Bromide; Ligation; Cloning

Findings

Chimeric gene construction is one of the most important procedures for molecular manipulation of genes and gene products. Today, with the advance of enzymes and cloning vectors and the assistance of polymerase chain reaction (PCR), the DNA segment can be inserted easily into a cloning vector without much ingenuity. The challenging aspect of gene construction is inserting multiple fragments into one vector. These multi-segment constructs are desirable since it is well documented that transgenes with inverted repeats are more potent in gene suppression than those of simple sense or anti-sense construct (Waterhouse et al. 1998; Wesley et al.2001; Hannon 2002; Watson et al. 2005). Current methods of chimeric gene construction involve preparing insert and vector with compatible ends (blunt ends or overhangs) and gel-purification of insert and vector for ligation. The gel-purification step normally uses ethidium bromide (EtBr) and UV radiation to identify bands of interest, which causes transformation efficiency to drop considerably (Flores et al. 1992). The newer and well-liked staining agent, SYBR Green (Molecular Probe, Eugene, OR), also requires UV radiation. Thus, one either performs a series of ligation steps by cloning the fragments into a vector one at a time or implements a multi-segment ligation, at the risk of obtaining inconsistent results. Another staining agent, methylene blue, does not require UV light for detection of DNA bands. Plasmid DNA prepared from methylene blue-stained gels showed a 2-fold higher transformation rate than that from EtBr-stained gels (Flores et al. 1992). However, methylene blue is much less sensitive than EtBr and is not commonly used.

Here I outline a simple method to isolate DNA without exposure to ethidium bromide (EtBr) and UV radiation for multi-segment cloning. Using these DNA segments, we consistently generated constructs of inverted RNA structures of leafy spurge (Euphorbia esula L.) genes encoding cullin-like protein, chlorophyll a/b-binding protein, and an unknown protein using three different cloning vectors: pUC19, pBlueScript-SK, and pBI121. All constructs involved ligation of four DNA segments simultaneously. Figure 1 illustrates an example of a multi-segment construct encoding an inverted cullin gene sequence.

Citation: Chao WS. Ethidium Bromide-Free DNA in Chimeric Gene Construction Involving Multiple DNA Segments. Austin J Plant Biol. 2015;1(1): 1002. ISSN:2472-3738