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Open Access Highly Accessed Methodology article

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

Chaokun Li1, Aiyun Wen1, Benchang Shen13, Jia Lu2, Yao Huang2 and Yongchang Chang1*

Author Affiliations

1 Division of Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ, USA

2 Department of Obstetrics and Gynecology; St. Joseph's Hospital and Medical Center, Phoenix, AZ, USA

3 Department of Genetics and Cell Biology, Guangzhou Medical University, Guangzhou, China

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BMC Biotechnology 2011, 11:92  doi:10.1186/1472-6750-11-92

Published: 12 October 2011

Abstract

Background

Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process.

Results

Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp.

Conclusion

Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.