Abstract
Megaprimer-based methodology has been widely applied in site-directed mutagenesis, but rarely used in gene splicing. In this article, we describe a modification of the megaprimer PCR method, which can efficiently create and amplify a specific ligated chimeric gene segment in a PCR reaction and under a common PCR program that is widely used by researchers. More importantly, this modified method for splicing two or more gene fragments together revealed the mechanism of the megaprimer PCR method, by elucidating the key factor in the megaprimer-based protocol. In this method, the denatured megaprimer divided into two strands. One strand was used as template DNA to regenerate megaprimer and the other strand was used as an oligonucleotide primer to create a ligated chimeric gene product. In this article, we detail the modified megaprimer protocol for creating and amplifying these chimeric gene products, including a specific protocol for large chimeric gene products. We also provide additional tips to increase specificity and efficiency of the protocols. In conclusion, the improved megaprimer PCR protocol is a simple, broadly applicable protocol for splicing two different gene fragments together without relying on restriction sites.
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Acknowledgments
The authors would gratefully acknowledge the laboratory support provided by Professor Xiaofang Luo. This work is supported by China National “948” Program (Grant No. 2005-4-34, 2007-4-02); National High Technology Program (2006AA10Z182); Special Research Fund for Doctor’s Degree Dissertation in Chinese Universities (20060022012); National Natural Science Foundation of China (30371148). This article has been edited by International Science Editing.
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Chen, JR., Lü, JJ. & Wang, HF. Rapid and Efficient Gene Splicing Using Megaprimer-Based Protocol. Mol Biotechnol 40, 224–230 (2008). https://doi.org/10.1007/s12033-008-9078-z
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DOI: https://doi.org/10.1007/s12033-008-9078-z