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Family shuffling, which generates chimeric progeny genes by recombining a set of naturally occurring homologous genes, is an extremely powerful approach for in vitro protein evolution. In comparison with other in vitro protein evolution methods, family shuffling has the advantage of sampling a larger portion of the sequence space that has been proven functionally rich by nature. So far, the most widely used technique to carry out family shuffling is DNA shuffling (1-5). However, one significant drawback associated with this method is the low frequency of chimeric genes (recombined gene products) in the shuffled library (6), which may be largely owing to the annealing of DNA fragments derived from the same parental genes (homo-duplex formation) whose probability is much higher than that of hetero-duplex formation. This is true even when the sequence homologies among parental genes are higher than 80% (7). [ABSTRACT FROM AUTHOR]