DNA shuffling

DNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment. It is used to rapidly increase DNA library size. DNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment. It is used to rapidly increase DNA library size. First, DNase is used to fragment a set of parent genes into pieces of 50-100 bp in length. This is then followed by a polymerase chain reaction (PCR) without primers- DNA fragments with sufficient overlapping homologous sequence will anneal to each other and are then extended by DNA polymerase. Several rounds of this PCR extension are allowed to occur, after some of the DNA molecules reach the size of the parental genes. These genes can then be amplified with another PCR, this time with the addition of primers that are designed to complement the ends of the strands. The primers may have additional sequences added to their 5' ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector. It is possible to recombine portions of these genes to generate hybrids or chimeric forms with unique properties, hence the term DNA shuffling.