Enzyme free cloning for high throughput gene cloning and expression.
Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.
de Jong
Rob N
RN
Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Daniëls
Mark A
MA
Kaptein
Rob
R
Folkers
Gert E
GE
D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases; D004798:Enzymes; D005822:Genetic Vectors; D023281:Genomics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004798:Enzymes; D011994:Recombinant Proteins; D011088:DNA Ligases