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US 6,440,668 B1Granted
Method of DNA shuffling with polynucleotides produced by blocking or interrupting a synthesis or amplification process
AssigneeDiversa Corporation
InventorJay M. Short
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 synthesis or amplification process to slow or halt synthesis or amplification of at least one polynucleotides, 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 polypeptides.
Claims12
An in vitro method for producing a polypeptide having an activity of interest, comprising:
- (a) 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, and/or polymerase inhibitors or poisons, wherein the member blocks or interrupts polynucleotide synthesis or amplification, thereby providing a plurality of polynucleotides at various stages of synthesis;
- (b) denaturing the plurality of polynucleotides obtained from step (b) to produce a mixture of single-stranded polynucleotides;
- (c) incubating the mixture of single stranded polynucleotides with a polymerase under conditions which result in annealing of the single stranded polynucleotides at regions of identity between the single-stranded polynucleotides and which results in the synthesis of at least one mutagenized double stranded polynucleotide;
- (d) repeating steps (c) and (d);
- (e) generating at least one mutagenized double stranded polynucleotide that encodes a polypeptide;
- and
- (f) screening the at least one mutagenized polynucleotide that encodes a polypeptide to determine the polypeptide that possesses an activity of interest.
4 more elements
A method for producing a polypeptide having an activity of interest, comprising:
- (a) blocking or interrupting a polynucleotide synthesis or amplification process in an isolated cell or cell free system with at least one member selected from the group consisting of UV light, one or more DNA adducts, DNA intercalating agents, and/or polymerase inhibitors or poisons, wherein the member blocks or interrupts polynucleotide synthesis or amplification, thereby providing a plurality of polynucleotides at various stages of synthesis;
- (b) denaturing the plurality of polynucleotides obtained from step (b) to produce a mixture of single-stranded polynucleotides;
- (c) incubating the mixture of single stranded polynucleotides with a polymerase under conditions which result in annealing of the single stranded polynucleotides at regions of identity between the single-stranded polynucleotides and which results in the synthesis of a mutagenized double stranded polynucleotide;
- (d) repeating steps (c) and (d);
- (e) cloning at least one mutagenized polynucleotide into an expression vector;
- (f) expressing the at least one mutagenized double stranded polynucleotide to obtain a polypeptide;
- and
- (g) screening the expressed polypeptide for an activity of interest.
5 more elements
A method of generating a bacteriophage display library, comprising:
- (a) blocking or interrupting a polynucleotide synthesis or amplification process in an isolated cell or cell free system with at least one member selected from the group consisting of UV light, one or more DNA adducts, DNA intercalating agents, and/or polymerase inhibitors or poisons, wherein the member blocks or interrupts polynucleotide synthesis or amplification, thereby providing a plurality of polynucleotides at various stages of synthesis;
- (b) denaturing the plurality of polynucleotides obtained from step (b) to produce a mixture of single-strand polynucleotides;
- (c) incubating the mixture of single stranded polynucleotides with a polymerase under conditions which result in annealing of the single stranded polynucleotides at regions of identity between the single-stranded polynucleotides and which results in the synthesis of a mutagenized double stranded polynucleotide;
- (d) repeating steps (b) and (c);
- (e) cloning at least one mutagenized polynucleotide into each of a plurality of bacteriophages to generate a bacteriophage display library;
- (f) expressing the mutagenized polynucleotides in the bacteriophage display library;
- and
- (g) screening the bacteriophage display library for a polypeptide of interest.
5 more elements
An in vitro method for producing a polypeptide having an activity of interest, comprising:
- (a) blocking or interrupting a polynucleotide synthesis or amplification process thereby providing a plurality of polynucleotides at various sages of synthesis;
- (b) denaturing the plurality of polynucleotides obtained from step (b) to produce a mixture of single-stranded polynucleotides;
- (c) incubating the mixture of single stranded polynucleotides with a polymerase under conditions which result in annealing of the single stranded polynucleotides at regions of identity between the single-stranded polynucleotides and which results in the synthesis of at least one mutagenized double stranded polynucleotide;
- (d) repeating steps (c) and (d);
- (e) generating at least one mutagenized double stranded polynucleotide that encodes a polypeptide;
- and
- (f) screening the at least one mutagenized polynucleotide that encodes a polypeptide to determine the polypeptide that possesses an activity of interest.
4 more elements
Dependencies are inferred from the claim text.