DNA SHUFFLING BY RANDOM FRAGMENTATION AND REASSEMBLY - IN-VITRO RECOMBINATION FOR MOLECULAR EVOLUTION

Computer simulations of the evolution of hmm sequences have demonstrat ed the importance of recombination of blocks of sequence rather than p oint mutagenesis alone. Repeated cycles of point mutagenesis, recombin ation, and selection should allow in vitro molecular evolution of comp lex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination i s reported. A 1-kb gene, after DNase I digestion and purification of 1 0- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassemble d. Complete recombination was obtained between two markers separated b y 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to th e fragment mixture and incorporated into the reassembled gene. Thus, m ixtures of synthetic oligonucleotides and PCR fragments can be mixed i nto a gene at defined positions based on homology. As an example, a li brary of chimeras of the human and murine genes for interleukin Ip has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with pare ntal DNA. The advantages of recombination over existing mutagenesis me thods are likely to increase with the numbers of cycles of molecular e volution.