Method of DNA shuffling with polynucleotides produced by blocking or interrupting a synthesis or amplification process
OwnerJay M. Shortinventor
Life of this patent
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Abstract
Disclosed is a process of performing “sexual” PCR which includes generating random polynucleotides by interrupting or blocking a synthesis or amplification process to show or halt synthesis or amplification of at least one polynucleotide, optionally amplifying the polynucleotides, and reannealing the polynucleotides to produce random mutant polynucleotides. Also provided are vector and expression vehicles including such mutant polynucleotides, polypeptides expressed by the mutant polynucleotides and a method for producing random mutant polypeptides.
Claims8
A method for producing mutant polynucleotides comprising:
- producing polynucleotides by blocking or interrupting a polynucleotide synthesis or amplification process with a member selected from the group consisting of UV light, one or more DNA adducts, DNA intercalating agents, DNA binding proteins, triple helix forming agents, competing transcription polymerase, chain terminators, and polymerase inhibitors or poisons, said member being capable of blocking or interrupting synthesis or amplification of a polynucleotide to provide a plurality of polynucleotides due to said polynucleotides being in various stages of synthesis or amplification, and
- subjecting said polynucleotides to an amplification procedure to amplify one or more of the polynucleotide or polynucleotides.
A process for producing mutant polynucleotides by a series of steps comprising:
- (a) producing oligonucleotides by blocking or interrupting a polynucleotide synthesis or amplification process with at least one member selected from the group consisting of UV light, one or more DNA adducts, DNA intercalating agents, chain terminators, and/or polymerase inhibitors or poisons, wherein said member is capable of blocking or interrupting polynucleotide synthesis or amplification and provide a plurality of polynucleotides due to their being in various stages of synthesis of amplification.
- (b) denaturing the resulting single or double stranded oligonucleotides to produce a mixture of single-stranded polynucleotides, optionally separating the polynucleotides into polls of polynucleotides having various lengths, and further optionally subjecting said polynucleotides to a priming and amplification procedure to amplify one or more oligonucleotides comprised by at least one of the polynucleotide pools;
- (c) incubating a plurality of said polynucleotides or at least one pool of said polynucleotides with a polymerase under conditions which result in annealing of said single-stranded polynucleotides at regions of identity between the single-stranded polynucleotides and formation of mutagenized double stranded polynucleotide chian;
- (d) repeating steps (c) and (d);
- (e) expressing at least one mutant polypeptide from said polynucleotide chain, or chains;
- and
- (f) screening said at least one mutant polypeptide for a useful activity.
Dependencies are inferred from the claim text.