Abstract
BackgroundRapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments.ResultsWe performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5–10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification.ConclusionsOur method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match.
Highlights
Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects
Overlap extension PCR was initially employed for fusion of two or three DNA fragments
The classical overlap extension PCR method generally consists of two steps and two separated reaction mixtures i.e. (1) In the overlap extension phase the outermost primers were no need and annealing temperature was relatively lower than the step (2) In the fused DNA amplifying phase, a new reaction mixture with the outermost primers was made and a new program was run at higher annealing temperature
Summary
Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. Former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments. Fusion of two or more DNA fragments is frequently required in many recombinant DNA projects but conventional restriction enzyme cloning and adapters cannot work well in many cases. Original overlap extension PCR, known as OE-PCR, was employed to splice two fragments precisely and quickly without the use of restriction enzymes [1,2,3]. Some modified OE-PCR methods can be applied in many areas, such as longlength DNA fusion [10,11,12], long-length gene cloning [13,14,15], gene mutation [16,17,18] and DNA circularization [19]. We modified OE-PCR, referred to simultaneous splicing overlap extension PCR (SSOE-PCR), which was allowed to
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