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Biol Proced Online. 2013 Sep 9;15(1):9. doi: 10.1186/1480-9222-15-9.

Simultaneous splicing of multiple DNA fragments in one PCR reaction.

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Hunan Provincial Key Laboratory of Phytohormones, Hunan Agricultural University, Changsha 410128, China.



Rapid 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.


We 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.


Our 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.

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