Random Chimeragenesis on Transient Templates

Random chimera genesis on transient templates (such as: " Random generation of mixed DNA using transient templates) ," short RACHITT, is a method to mutagenize enzymes by means of recombination events in the sense of directed evolution.

Operation

The method allows for the recombination of homologous genes from different organisms. To the gene to be cut by means of a deoxyribonuclease and separated the resulting fragments by length. The fragments of desired length can be hybridized with a gene homologous to the starting RNA backbone, did not hybridize, protruding ends are then cut away, and the gaps between the fragments by a DNA polymerase and a DNA ligase is filled.

The RNA backbone is then removed by a ribonuclease, and multiplies the recombinant DNA for the next use by PCR. Compared to other methods such as DNA shuffling flows at a RACHITT hardly a background of the origin of genes used ( unshuffled background) in the further work, since the used RNA scaffold is degraded prior to PCR.