Abstract
BackgroundWith the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired.ResultsA bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3′ end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/μg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs).ConclusionsWe describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.
Highlights
With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated
To examine if insertion of control of cell death B (ccdB) gene in the reverse orientation would lead to cell lethality, both pXST and pXST-R were transformed into E. coli DB3.1 and DH5α, and the transformants were grew on LB plates with ampicillin selection independently
DH5α with pXST-R can grow on the LB-Amp plate with adding IPTG (Fig. 3d), which indicated that the insertion of ccdB in the reverse orientation cannot be expressed, cannot result in lethality
Summary
With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. Due to the high cost and complicated protocol, hardly can commercially available ligase-free methods meet the requirement for large scale cloning and be used in most. The cohesive-end ligation links the target DNA fragments (inserts) to plasmids through complementary between the inserts and plasmid vectors, resulting in high recombinant efficiency. The blunt-end cloning walks around such problems by avoiding the digestion of the insert fragments. New problems arise for the blunt-end cloning because the blunt ends of vectors could be self-ligated, decreasing the available vectors for ligating inserts. TA cloning is the simplest and most efficient approach for cloning of PCR products, because the method avoid the requirement of digesting the insert fragments and the self-ligation problem of vectors
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