Improvement of TA Cloning Method to Facilitate Direct Directional Cloning of PCR Products
Year: 2014
Type: article
Abstract: Directional cloning is a prerequisite for the construction of expression vectors in molecular biology laboratories. Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional. Here we reported a novel PCR products cloning vector with one deoxythymidine overhang and one deoxycytidine overhang at two 3'-ends respectively. With the choice of nucleotides of 5'-ends of PCR primers, PCR products can be cloned to this vector both directly and directionally. The feasibility and efficacy of this cloning method were confirmed by using a pET-17b derivative vector and a green fluorescent protein gene (EGFP) and a red fluorescent protein reporter (Ds-Red) gene. This cloning strategy may be useful in the high-throughput construction of expression vectors and could be viewed as an interesting improvement of existing TA cloning method.
Source: Applied Mechanics and Materials
Authors: Ji Gang Li, Guo Ying Han, Xiu Min Li, Jiao Jiao Sun, Ke Jing Song, Ting Zhang
Institution: Hebei University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
OpenAlex ID: W1985481886