Cloning short DNA into plasmids by one-step PCR(1)
Several PCR-based cloning protocols have been proposed to skip the use of DNA ligases and restriction endonucleases. DNA inserts below 150 bp can be directly synthesized, but it is cost prohibitive with limited productivity. Also, the effects of synthesizing two short oligonucleotides below 150 bp still depend on the efficiency of annealing, endonuclease, and ligase, etc.
Compared with the traditional construction method the schematic map of our method is illustrated. PCR products were absent in the negative control, while the pEGFP-N1-HA reaction system showed a 4.7 kb PCR product.
The HA-tag expression in 293T cells was detected by western blotting. HA-tag was successfully expressed in cells. The HA-GFP fusion protein was clearly detected by confocal microscopy.
Seamless ligation cloning extract (SLiCE) method is a commonly used method, which utilizes bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. The DNA fragment of interest can be inserted at any position other than the promoter in this new method. This one-step PCR method synthesized 89 bp DNA, about 47% less than 131 bp of the In-Fusion cloning. The efficiency may not be constant because the plasmid template used in PCR amplification has some transformation potential. The DpnI digestion time of the PCR products plays key roles in the success of this method.