DNA shuffling

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DNA shuffling, also known as sexual PCR (in reference to polymerase chain reaction, or PCR), is a way to rapidly propagate beneficial mutations in a directed evolution experiment. It is used to rapidly increase DNA library size.

DNA shuffling, in contrast, is PCR without synthetic primers. In this process, a family of related genes--say, the ones that codes for the surface proteins of three different HIV isolates--are first chopped up with enzymes. The gene fragments then are heated up to separate them into single-stranded templates. Some of these fragments will bind to other fragments that share complementary DNA regions, which in some cases will be from other family members. Regions of DNA that are non-complementary hang over the ends of the templates (see illustration). The PCR reaction then treats the complementary regions as primers and builds the new double-helical DNA. But PCR also adds bases to the overhanging piece of the primer, forming a double helix there, too. This ultimately creates a mixed structure or chimera. In the final step, PCR reassembles these chimeras into full-length, shuffled genes.

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