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pXST, a novel vector for TA cloning and blunt-end cloning

BACKGROUND: With 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. RESULTS: A bifunctional vector pXST has been construc...

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Autores principales: Liu, Qin, Dang, Hui-Jie, Wu, Yuan-Hang, Li, Min, Chen, Yin-Hua, Niu, Xiao-Lei, Li, Kai-Mian, Luo, Li-Juan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045858/
https://www.ncbi.nlm.nih.gov/pubmed/30005664
http://dx.doi.org/10.1186/s12896-018-0456-8
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author Liu, Qin
Dang, Hui-Jie
Wu, Yuan-Hang
Li, Min
Chen, Yin-Hua
Niu, Xiao-Lei
Li, Kai-Mian
Luo, Li-Juan
author_facet Liu, Qin
Dang, Hui-Jie
Wu, Yuan-Hang
Li, Min
Chen, Yin-Hua
Niu, Xiao-Lei
Li, Kai-Mian
Luo, Li-Juan
author_sort Liu, Qin
collection PubMed
description BACKGROUND: With 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. RESULTS: A 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 × 10(6) 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). CONCLUSIONS: We 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.
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spelling pubmed-60458582018-07-16 pXST, a novel vector for TA cloning and blunt-end cloning Liu, Qin Dang, Hui-Jie Wu, Yuan-Hang Li, Min Chen, Yin-Hua Niu, Xiao-Lei Li, Kai-Mian Luo, Li-Juan BMC Biotechnol Methodology Article BACKGROUND: With 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. RESULTS: A 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 × 10(6) 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). CONCLUSIONS: We 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. BioMed Central 2018-07-13 /pmc/articles/PMC6045858/ /pubmed/30005664 http://dx.doi.org/10.1186/s12896-018-0456-8 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Liu, Qin
Dang, Hui-Jie
Wu, Yuan-Hang
Li, Min
Chen, Yin-Hua
Niu, Xiao-Lei
Li, Kai-Mian
Luo, Li-Juan
pXST, a novel vector for TA cloning and blunt-end cloning
title pXST, a novel vector for TA cloning and blunt-end cloning
title_full pXST, a novel vector for TA cloning and blunt-end cloning
title_fullStr pXST, a novel vector for TA cloning and blunt-end cloning
title_full_unstemmed pXST, a novel vector for TA cloning and blunt-end cloning
title_short pXST, a novel vector for TA cloning and blunt-end cloning
title_sort pxst, a novel vector for ta cloning and blunt-end cloning
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045858/
https://www.ncbi.nlm.nih.gov/pubmed/30005664
http://dx.doi.org/10.1186/s12896-018-0456-8
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