title: Rapid restriction enzyme-free cloning of PCR products: a high-throughput method applicable for library construction.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Chaudhary
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Nimisha
authors.initials: N
authors.lastname: Shrivastava
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Vaishali
authors.initials: V
authors.lastname: Verma
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Shilpi
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Charanpreet
authors.initials: C
authors.lastname: Kaur
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Payal
authors.initials: P
authors.lastname: Grover
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
authors.forename: Amita
authors.initials: A
authors.lastname: Gupta
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D057166:High-Throughput Screening Assays / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004262:DNA Restriction Enzymes