Diagrammatic representation of DNA shuffling is shown in Figure 5.

Figure 5: Diagrammatic representation of DNA shuffling

In each PCR cycle repeat the average fragment length increases. After many cycles of PCR without adding primers, molecules of original size are expected. Recombination occurs when a template from one molecule primes a fragment from another molecule.

The purpose of DNA shuffling is to recombine beneficial mutations from different molecules, and obtain molecules with even increased function. Further DNA shuffling improves the search of local fold space by means of a random yet correlated combination of homologous coding fragments that contain limited numbers of beneficial amino acid substitutions.