Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.
Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.|Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
Hui
H
Zhang
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
Chang-Jun
CJ
Liu
Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China.
Hui
H
Jiang
Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
Lu
L
Zhou
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
Wen-Ying
WY
Li
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
Ling-Yun
LY
Zhu
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China.
Lei
L
Wu
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China dongyizhang@nudt.edu.cn er_meng@qq.com.
Er
E
Meng
Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, Hunan 410073, China dongyizhang@nudt.edu.cn er_meng@qq.com.
Dong-Yi
DY
Zhang
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results
D017931:DNA Primers; D004247:DNA
DNA insertion; Mega Primer; Seamless cloning