Document Detail

Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system.
MedLine Citation:
PMID:  14736459     Owner:  NLM     Status:  MEDLINE    
Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions. In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined. In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector, various combinations of attB and attP pairs were tested for their reactivities in recombination. The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3, attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system. Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown.
Yukari Sasaki; Takefumi Sone; Shouhei Yoshida; Kazuhide Yahata; Junko Hotta; Jonathan D Chesnut; Takeshi Honda; Fumio Imamoto
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of biotechnology     Volume:  107     ISSN:  0168-1656     ISO Abbreviation:  J. Biotechnol.     Publication Date:  2004 Feb 
Date Detail:
Created Date:  2004-01-22     Completed Date:  2004-08-31     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  233-43     Citation Subset:  IM    
Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka Suita, Osaka 565-0871, Japan.
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MeSH Terms
Bacterial Proteins
Base Sequence
Cloning, Molecular* / methods
DNA / genetics*
Escherichia coli / genetics
Genetic Vectors
Green Fluorescent Proteins
Luminescent Proteins / genetics
Lysogeny / genetics
Molecular Sequence Data
Recombinant Proteins / genetics
Recombination, Genetic*
Transformation, Bacterial
Reg. No./Substance:
0/Bacterial Proteins; 0/Luminescent Proteins; 0/Recombinant Proteins; 147336-22-9/Green Fluorescent Proteins; 9007-49-2/DNA; EC 2.7.7.-/Integrases

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