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Alternative Seamless Cloning Strategies in Fusing Gene Fragments Based on Overlap-PCR

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Abstract

Gene fragment swapping and site-directed mutagenesis are commonly required in dissecting functions of gene domains. While there are many approaches for seamless fusion of different gene fragments, new methods are yet to be developed to offer higher efficiency, better simplicity, and more affordability. In this study, we showed that in most cases overlap-PCR was highly effective in creating site-directed mutagenesis, gene fragment deletion, and substitutions using RUS1 and RUS2 as example. While for cases where the overlap-PCR approach is not feasible due to complex secondary structure of gene fragments, a unique restriction site can be generated at the overlapped region of the primers through synonymous mutations. Then different gene fragments can be seamlessly fused through traditional restriction digestion and subsequent ligation. In conclusion, while the classical overlap-PCR is not feasible, the modified overlap-PCR approaches can provide effective and alternative ways to seamlessly fuse different gene fragments.

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Abbreviations

Cab:

Chlorophyll a/b binding protein

CTP:

Chloroplast transit peptide

CRISPR/Cas9:

Clustered regularly interspaced short palindromic repeats/ CRISPR-associated 9

DUF647:

Domain of unknown function 647

IDP:

Intrinsically disordered protein

IFPC:

Inverse Fusion PCR Cloning

iVEC:

In vivo Escherichia coli cloning

OEPRC:

Overlap extension PCR and recombination cloning

ORF:

Open reading frame

RUS :

ROOT UVB SENSITIVE

SLiCE:

Seamless ligation cloning extract

TOCC:

Tocopherol cyclase

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Funding

XWH acknowledges the financial support from the Natural Science Foundation of China (Grant No. 30971709). Funding body was not involved in the design of the study; collection, analysis, interpretation of data, and in writing the manuscript.

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Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China

    Xue-Wen Hou

  2. Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA, 94132, USA

    Hong-Yun Tong & Zheng-Hui He

Authors
  1. Xue-Wen Hou
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  2. Hong-Yun Tong
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Contributions

XWH, HYT, and ZHH planned and designed this research. XWH and HYT performed experiments. XWH analyzed the data and wrote the manuscript. ZHH edited the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Xue-Wen Hou.

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Hou, XW., Tong, HY. & He, ZH. Alternative Seamless Cloning Strategies in Fusing Gene Fragments Based on Overlap-PCR. Mol Biotechnol 63, 221–231 (2021). https://doi.org/10.1007/s12033-020-00298-0

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