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Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

ABSTRACT: 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.

SUBMITTER: Zhang H

PROVIDER: S-EPMC5333777 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

Zhang Hui H Liu Chang-Jun CJ Jiang Hui H Zhou Lu L Li Wen-Ying WY Zhu Ling-Yun LY Wu Lei L Meng Er E Zhang Dong-Yi DY

Bioscience reports 20170302 2

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 ...[more]

PMID: 28183872

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