Tfu Direct™ site-directed mutagenesis system is an inverse PCR based technique which enables site-specific mutation in almost any plasmid template, thus avoiding the need for subcloning into bacteriophage based vectors and the subsequent rescue of ssDNA. Tfu Direct™ provides a simple and rapid method for the successful production of point mutations, deletions or insertions in virtually any double stranded plasmid (see figure).
Tfu Direct™ uses our high-fidelity Tfu polymerase and a reduced cycling number to decrease the possibility of spurious mutations during the PCR step of the procedure. The PCR products are digested with the endonuclease Dpn I. Dpn I (recognition site 5'- Gm6ATC-3') is specific for methylated and hemimethylated DNA which applies to any DNA isolated from almost all Escherichia coli strains. Any plasmid generated by PCR however, is non-methylated and therefore resistant to digestion by Dpn I. This exact difference between the two plasmid populations at the end of the PCR step offers a method for removing all the original, non-mutated template plasmid DNA from the reaction. The remaining mixture is then purified, ligated and transformed into competent E. coli cells.
Applications : point mutations, insertions and deletions in any plasmid borne DNA sequence.
The Tfu Direct™ site-directed mutagenesis kit provides all the reagents necessary to perform 25 mutagenesis reactions.
The kit has three separate parts, one box to be stored at -20°C, one at -80°C with three components to be stored at RT/4°C.