Abstract
BackgroundInvitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gateway destination vectors have been created while MultiSite Gateway is limited to few destination vectors and therefore to few applications. The aim of this work was to make the MultiSite Gateway technology available for multiple biological purposes.ResultsWe created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original destination vectors can still be utilized for promoter-gene or translational fusions after the conversion.ConclusionOur tool makes MultiSite Gateway a more widely accessible technology and expands its applications by exploiting all the features of the Gateway vectors already available.
Highlights
Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors
We decided to take advantage of the large number of Gateway single-site destination vectors and developed a new method to convert them to MultiSite Gateway
The functional part of MultiSite Gateway destination vectors is the attR4-attR3 cassette which can recombine with three Gateway donor vectors carrying the appropriate attL sites (Fig 2B) [7]
Summary
Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. Recombinase-based cloning technologies are becoming increasingly popular because of their easy use and high efficiency These tools exploit bacterial or viral site-specific recombinases like the bacteriophage P1 Cre, the Saccharomyces cerevisiae FLP or the bacteriophage lambda integrase [1,2,3]. Gateway (Invitrogen) is one of the most popular recombination cloning technologies [3] It is based on the Escherichia coli bacteriophage lambda integrase/att system [5]. The BP reaction is catalyzed by the BP Clonase enzyme mix (Invitrogen), which recombines attB sites with attP sites. The resulting construct can be selected using a com-
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