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.
Highlights
The availability of genome sequences from a large number of organisms has created a wide-spread need to clone complete sets of open reading frames (ORFs) followed by the expression and purification of the encoded gene products in a high-throughput manner to examine the functions of genes and proteins in organisms
We demonstrate the strategy is effective for cloning inserts of various sizes (0.5 kbp to 2.2 kbp) in single tube format suitable for high-throughput applications, and for high efficiency cloning during the construction of genome-scale libraries
Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 59-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 baselong overhangs
Summary
The availability of genome sequences from a large number of organisms has created a wide-spread need to clone complete sets of open reading frames (ORFs) followed by the expression and purification of the encoded gene products in a high-throughput manner to examine the functions of genes and proteins in organisms. An ideal high-throughput cloning method should include a minimal number of steps without the need for intermediate purification and preferably be performed in the same tube in which the gene-of interest was amplified. For such cloning, (i) the reagents used to prepare the vector and insert should not be proprietary and should be available from multiple sources; (ii) the vector should not self-ligate and the insert should not form concatemers, rather ligate to the vector in the desired orientation; (iii) the primers for amplification should carry only small additional sequences without the need for any modifications, and thereby only add a sequence encoding a few amino acids at the vector/insert junctions to serve as spacers, and ; (iv) the vector design should be amenable to modifications such as incorporation of different tags to enable purification and/or enhance solubility, or changing the promoter, antibiotic selection marker and even the origin of replication
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