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. because it is a recombination between different DNA species with different mutations.

Procedure

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 portion of these genes to generate hybrids or chimeric forms with unique properties, which is called DNA shuffling.

Shuffling methods

Using restriction enzymes

1. Restriction enzymes that cut in similar places are used to digest members of the gene family
2. DNA fragments are joined together with DNA ligase
3. Large numbers of hybrids that can be tested for unique properties are produced

Using DNAse 1

1. Different members of the gene family are fragmented using DNase 1 followed by PCR
2. During PCR different members of the family are cross-primed, DNA fragments with high homology will anneal to each other
3. The generated hybrids are then used to generate a library of mutants, which are tested for unique properties

See also

• SCOPE (protein engineering)
• RACHITT
• Homologous recombination