Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis

Traditional cut-paste DNA cloning is often limited by the availability of restriction enzyme sites. Here, we described the complementary annealing mediated by exonuclease (CAME), in which the insert or vector fragment is amplified to carry sequences complementary to the other, and both fragments are...

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Autores principales: Sun, Shuhui, Huang, Hao, Qi, Yingchuan Billy, Qiu, Mengsheng, Dai, Zhong-Min
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2015
Materias:
Article; Medical Biotechnology
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686905/
https://www.ncbi.nlm.nih.gov/pubmed/26740790
http://dx.doi.org/10.1080/13102818.2014.988094
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author Sun, Shuhui
Huang, Hao
Qi, Yingchuan Billy
Qiu, Mengsheng
Dai, Zhong-Min
author_facet Sun, Shuhui
Huang, Hao
Qi, Yingchuan Billy
Qiu, Mengsheng
Dai, Zhong-Min
author_sort Sun, Shuhui
collection PubMed
description Traditional cut-paste DNA cloning is often limited by the availability of restriction enzyme sites. Here, we described the complementary annealing mediated by exonuclease (CAME), in which the insert or vector fragment is amplified to carry sequences complementary to the other, and both fragments are modified by exonuleases to create directional single-stranded overhangs. The two recessed DNA fragments are joined through complementary strand annealing. The CAME is highly efficient for cloning the DNA of at least 12 kb and single DNA fragment out of a complex DNA sample. Moreover, the application of CAME greatly improved the efficiency of site-directed mutagenesis.
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spelling pubmed-46869052016-01-04 Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis Sun, Shuhui Huang, Hao Qi, Yingchuan Billy Qiu, Mengsheng Dai, Zhong-Min Biotechnol Biotechnol Equip Article; Medical Biotechnology Traditional cut-paste DNA cloning is often limited by the availability of restriction enzyme sites. Here, we described the complementary annealing mediated by exonuclease (CAME), in which the insert or vector fragment is amplified to carry sequences complementary to the other, and both fragments are modified by exonuleases to create directional single-stranded overhangs. The two recessed DNA fragments are joined through complementary strand annealing. The CAME is highly efficient for cloning the DNA of at least 12 kb and single DNA fragment out of a complex DNA sample. Moreover, the application of CAME greatly improved the efficiency of site-directed mutagenesis. Taylor & Francis 2015-01-02 2014-12-10 /pmc/articles/PMC4686905/ /pubmed/26740790 http://dx.doi.org/10.1080/13102818.2014.988094 Text en © 2014 The Author(s). Published by Taylor & Francis. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
spellingShingle Article; Medical Biotechnology
Sun, Shuhui
Huang, Hao
Qi, Yingchuan Billy
Qiu, Mengsheng
Dai, Zhong-Min
Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
title Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
title_full Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
title_fullStr Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
title_full_unstemmed Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
title_short Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
title_sort complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis
topic Article; Medical Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686905/
https://www.ncbi.nlm.nih.gov/pubmed/26740790
http://dx.doi.org/10.1080/13102818.2014.988094
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