Random Chimeragenesis on Transient Templates

Random chimeragenesis on transient templates (for example: "Random generation of mixed DNA with the help of transient templates)", RACHITT for short, is a method to mutagenize enzymes with the help of recombination processes in the sense of directed evolution .

functionality

The method enables the recombination of homologous genes from different organisms. For this purpose, the genes are cut with the help of a deoxyribonuclease and the resulting fragments are separated according to length. The fragments of the desired length are hybridized with an RNA framework homologous to the starting genes , unhybridized, protruding ends are then cut away and the gaps between the fragments are filled with a DNA polymerase and a DNA ligase .

The RNA scaffold is then broken down by a ribonuclease and the recombined DNA is amplified by means of PCR for subsequent use . In contrast to other methods such as DNA shuffling , RACHITT hardly uses any background of the original genes used ( unshuffled background ) in further work, as the RNA framework used is broken down before the PCR.

Web links

• Comparison of DNA shuffling and RACHITT at Nature Biosciences

swell

• Wayne M. Coco, William E. Levinson, Michael J. Crist, Harm J. Hektor, Aldis Darzins, Philip T. Pienkos, Charles H. Squires, Daniel J. Monticello: DNA shuffling method for generating highly recombined genes and evolved enzymes . In: Nature Biotechnology . tape 19 , no. 4 , 2001, p. 354-359 (English).