Molecular Evolution Platform used for Taq polymerase

Technology based on site directed mutagenesis of the gene for Taq polymerase and then the selection of the mutated enzyme by its resistance to pressure imposed

KAPA has developed a new generation of Taq polymerase with its platform of directed molecular evolution.
The pressure varies according to objective (robsutesse, speed ...)



Principle of technology

1. Cloning of the Taq polymerase 2a. random mutagenesis 2b. Cloning of mutants in E. coli 2c. Production of the mutated Taq polymerase 3a. Resuspension of E. Coli in soluble phase on a hydrophobic phase 3b. emulsion 4a. PCR under certain pressure conditions: amplification of mutant clones selected 4b. Visualization of the amplification obtained

1 - Each bacterium contains a plasmid encoding a mutant wild type Taq and is resuspended in a medium containing the primers amplifying the mutated Taq 2 - Each bacterium is isolated by emulsion in a bubble containing medium-soluble 3 - PCR in the presence of a pressure condition (destruction of the bacteria by heat) 4 - Amplification of the amplicons encoding a Taq resistant pressure conditions 5 - Merging all the drops to get the starting soluble medium 6 - Sequencing of mutant candidates for a second round of selection

  - Addition of  NaCl in the soluble medium

   - Addition of  urea in the soluble medium

   - Addition of  SDS in the soluble medium

   - Addition of  ethanol in the soluble medium

   - Stop of elongation after 3 secondes

  - Addition of  polyphenols in the soluble medium

   - Addition of  SYBR Green in the soluble medium

 Tell a friend