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The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly

Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro, including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel me...

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Detalles Bibliográficos
Autores principales: Chen, Wei-Hua, Qin, Zhong-Jun, Wang, Jin, Zhao, Guo-Ping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632120/
https://www.ncbi.nlm.nih.gov/pubmed/23444142
http://dx.doi.org/10.1093/nar/gkt122
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author Chen, Wei-Hua
Qin, Zhong-Jun
Wang, Jin
Zhao, Guo-Ping
author_facet Chen, Wei-Hua
Qin, Zhong-Jun
Wang, Jin
Zhao, Guo-Ping
author_sort Chen, Wei-Hua
collection PubMed
description Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro, including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated ‘MASTER Ligation’, by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, (m)CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (∼29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F.
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spelling pubmed-36321202013-04-22 The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly Chen, Wei-Hua Qin, Zhong-Jun Wang, Jin Zhao, Guo-Ping Nucleic Acids Res Methods Online Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro, including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated ‘MASTER Ligation’, by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, (m)CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (∼29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F. Oxford University Press 2013-04 2013-02-26 /pmc/articles/PMC3632120/ /pubmed/23444142 http://dx.doi.org/10.1093/nar/gkt122 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Chen, Wei-Hua
Qin, Zhong-Jun
Wang, Jin
Zhao, Guo-Ping
The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
title The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
title_full The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
title_fullStr The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
title_full_unstemmed The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
title_short The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
title_sort master (methylation-assisted tailorable ends rational) ligation method for seamless dna assembly
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632120/
https://www.ncbi.nlm.nih.gov/pubmed/23444142
http://dx.doi.org/10.1093/nar/gkt122
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