Abstract
Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.
Highlights
As a foundational technology in molecular biology, plasmid construction is essential for investigating the functions of DNA and protein [1,2]
We find that almost all methods construct nc-plasmid DNAs, such as type II cloning, Gibson [27], SLiCE [25], the DNA amplification methods [4,6,9,10,11], the ExnaseII cloning, and the intra-homology recombination (HR)
We develop an intra-HR to amplify plasmid DNAs containing homology 5 - and 3 -terminus
Summary
As a foundational technology in molecular biology, plasmid construction is essential for investigating the functions of DNA and protein [1,2]. Many cloning methods have been reported, plasmid construction compromises transformant efficiency (i.e., number of transformants per ng of plasmid DNAs used in transformation) with plasmid accuracy (i.e., rate of scarless plasmids). The type II cloning method is limited by low activity of restriction enzymes, insertion of restriction sites, and specific positions for insertion [3]. These drawbacks drive DNA amplification methods to serve amplified inserts as megaprimers [2,4,5,6,7,8]. Efficient for creating over 10 transformants/ng, the method is compromised by constructing occasionally scar plasmids, i.e., insert-vector junctions contain nucleotide deletion, insertion, or mutation. Scar junction is a general problem in plasmid construction [4,5,7,9,10,11,12,13,14], and a frame-shift scar makes functional analysis impossible
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
