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Rapid Restriction Enzyme-Free Cloning Of PCR Products: A High-Throughput Method Applicable For Library Construction

V. Chaudhary, Nimisha Shrivastava, V. Verma, Shilpi Das, Charanpreet Kaur, Payal Grover, Amita Gupta
Published 2014 · Biology, Medicine

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Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatment. The BsaI sites in the vector are oriented in such a manner that upon digestion with BsaI, a stuffer sequence along with both BsaI recognition sequences is removed. The sequence of the four base-long overhangs produced by BsaI cleavage were designed to be non-palindromic, non-compatible to each other. Therefore, only ligation of an insert carrying compatible ends allows directional cloning of the insert to the vector to generate a recombinant without recreating the BsaI sites. We also developed rapid protocols for insert preparation and cloning, by which the entire process from PCR to transformation can be completed in 6–8 h and DNA fragments ranging in size from 200 to 2200 bp can be cloned with equal efficiencies. One protocol uses a single tube for insert preparation if amplification is performed using polymerases with low 3′-exonuclease activity. The other protocol is compatible with any thermostable polymerase, including those with high 3′-exonuclease activity, and does not significantly increase the time required for cloning. The suitability of this method for high-throughput cloning was demonstrated by cloning batches of 24 PCR products with nearly 100% efficiency. The cloning strategy is also suitable for high efficiency cloning and was used to construct large libraries comprising more than 108 clones/µg vector. Additionally, based on this strategy, a variety of vectors were constructed for the expression of proteins in E. coli, enabling large number of different clones to be rapidly generated.
This paper references
Construction of biologically functional bacterial plasmids in vitro.
S. Cohen (1973)
Use of T7 RNA polymerase to direct expression of cloned genes.
F. Studier (1990)
Ligation-independent cloning of PCR products (LIC-PCR).
C. Aslanidis (1990)
An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase.
P. Chowdhury (1994)
PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.
J. Cline (1996)
GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes.
A. J. Walhout (2000)
Universal TA cloning.
M. Zhou (2000)
DNA cloning using in vitro site-specific recombination.
J. Hartley (2000)
Protein production by auto-induction in high density shaking cultures.
F. W. Studier (2005)
Genome sequencing in microfabricated high-density picolitre reactors
M. Margulies (2005)
Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites
K. Colwill (2006)
A universal cloning vector using vaccinia topoisomerase I
L. Geng (2006)
A One Pot, One Step, Precision Cloning Method with High Throughput Capability
Carola Engler (2008)
A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme.
Ippei Kotera (2008)
Killing activity and rescue function of genome-wide toxin-antitoxin loci of Mycobacterium tuberculosis.
Amita Gupta (2009)
The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning.
N. Berrow (2009)
GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules
A. Sarrión-Perdigones (2011)
A Modular Cloning System for Standardized Assembly of Multigene Constructs
E. Weber (2011)
One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies
Jae-Yeon Jeong (2012)
Intraviral protein interactions of Chandipura virus
K. Kumar (2012)
SLiCE: a novel bacterial cell extract-based DNA cloning method
Y. Zhang (2012)
A Novel Helper Phage Enabling Construction of Genome-Scale ORF-Enriched Phage Display Libraries
A. Gupta (2013)
Rapid Restriction Enzyme-Free PCR Product Cloning PLOS ONE | www.plosone

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