GENE SHUFFLING METHODS

摘要

Disclosed methods pertain to nucleic acid shuffling techniques that employ repeated short extension cycles. In each such cycle, strand extension along a template fragment is limited such that the strand extends only for a relatively short length (e.g., a few base pairs). Repeated short extension cycles cause many template switches during shuffling and thereby produce chimeric products with many crossovers. The methods may employ a pre-shuffling truncation or excision operation in which one or more parent nucleic acids has a portion of its full-length sequence truncated or excised. Shuffling with truncated parent nucleic acids introduces crossovers at the location of the truncation. Apparatus for implementing the disclosed methods may include appropriately configured thermocycling tools.

主权项

1. A method of conducting nucleic acid recombination to facilitate incorporation of crossovers in variant sequences, the method comprising:
(a) combining fragments of two or more parent nucleic acids; (b) annealing single stranded fragments from the two or more parent nucleic acids to produce annealed single stranded fragments, wherein at least some of the annealed single stranded fragments have overhanging single stranded portions attached to a double stranded portion; (c) incompletely extending the annealed single stranded fragments to produce incompletely extended single stranded fragments, wherein, on average across the annealed fragments from the two or more parent nucleic acids, the extension is not more than about 50% of the overhanging single stranded portion of the annealed single stranded fragments existing prior to extension; (d) denaturing the incompletely extended single stranded fragments produced in (c); and (e) repeating (b)-(d) at least about 5 times to produce variant sequences, wherein the repetitions of (b) comprise annealing the incompletely extended single stranded fragments from (c).