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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
10.1073/PNAS.70.11.3240
Construction of biologically functional bacterial plasmids in vitro.
S. Cohen (1973)
10.1016/0076-6879(90)85008-C
Use of T7 RNA polymerase to direct expression of cloned genes.
F. Studier (1990)
10.1093/NAR/18.20.6069
Ligation-independent cloning of PCR products (LIC-PCR).
C. Aslanidis (1990)
10.1006/PREP.1994.1013
An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase.
P. Chowdhury (1994)
10.1093/NAR/24.18.3546
PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.
J. Cline (1996)
10.1016/S0076-6879(00)28419-X
GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes.
A. J. Walhout (2000)
10.21775/cimb.002.001
Universal TA cloning.
M. Zhou (2000)
10.1101/GR.143000
DNA cloning using in vitro site-specific recombination.
J. Hartley (2000)
10.1016/J.PEP.2005.01.016
Protein production by auto-induction in high density shaking cultures.
F. W. Studier (2005)
10.1038/nature03959
Genome sequencing in microfabricated high-density picolitre reactors
M. Margulies (2005)
10.1186/1472-6750-6-13
Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites
K. Colwill (2006)
10.1385/MB:33:1:23
A universal cloning vector using vaccinia topoisomerase I
L. Geng (2006)
10.1371/journal.pone.0003647
A One Pot, One Step, Precision Cloning Method with High Throughput Capability
Carola Engler (2008)
10.1016/j.jbiotec.2008.07.1816
A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme.
Ippei Kotera (2008)
10.1111/j.1574-6968.2008.01400.x
Killing activity and rescue function of genome-wide toxin-antitoxin loci of Mycobacterium tuberculosis.
Amita Gupta (2009)
10.1007/978-1-59745-196-3_5
The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning.
N. Berrow (2009)
10.1371/journal.pone.0021622
GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules
A. Sarrión-Perdigones (2011)
10.1371/journal.pone.0016765
A Modular Cloning System for Standardized Assembly of Multigene Constructs
E. Weber (2011)
10.1128/AEM.00844-12
One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies
Jae-Yeon Jeong (2012)
10.1007/s00705-012-1389-5
Intraviral protein interactions of Chandipura virus
K. Kumar (2012)
10.1093/nar/gkr1288
SLiCE: a novel bacterial cell extract-based DNA cloning method
Y. Zhang (2012)
10.1371/journal.pone.0075212
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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