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Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries

Nature Protocols volume 6, pages 242–251 (2011)Cite this article

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Abstract

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.

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Figure 1: Schematic diagram of CPEC cloning of combinatorial gene libraries.
Figure 2: Schematic diagrams of primer design in CPEC.
Figure 3: CPEC cloning of the lacZα gene library.
Figure 4: CPEC cloning of combinatoral gene libraries.

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Acknowledgements

We thank Z. Chen and P. Zuo for technical assistance. J.T. is a Beckman Young Investigator and a recipient of a Hartwell Individual Biomedical Research Award.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

    Jiayuan Quan & Jingdong Tian

  2. Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA

    Jiayuan Quan & Jingdong Tian

Authors
  1. Jiayuan Quan
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  2. Jingdong Tian
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Contributions

J.T. conceived the CPEC strategies and supervised the project. J.Q. performed the experiments. Both authors contributed extensively to the experimental design, protocol development and manuscript preparation.

Corresponding author

Correspondence to Jingdong Tian.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

PCR templates and primers used in the anticipated result examples. (DOC 28 kb)

Supplementary Table 2

Primer sequences used in the Anticipated Result Section. (DOC 31 kb)

Supplementary Figure 1

CPEC cloning of 14 gene libraries in parallel. The upper bands (red arrow) are the full-length cloning products whereas the lower bands (blue arrow) are the unincorporated vectors. Five thermal cycles were performed in CPEC. (DOC 361 kb)

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Quan, J., Tian, J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc 6, 242–251 (2011). https://doi.org/10.1038/nprot.2010.181

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