FOCUSED LIBRARY GENERATION
First Claim
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1. A method of producing a library of mutated nucleic acids, said method comprising:
- providing a single-stranded template nucleic acid comprising a first sequence of nucleotides;
providing a first set of primers, wherein a majority of said first set of primers are perfectly complementary to at least a portion of said first sequence of nucleotides;
providing a second set of primers, wherein a majority of said second set of primers are complementary to at least a portion of said first sequence of nucleotides except for at least one pre-selected mismatched nucleotide complementary to at least one targeted mutation;
combining said template nucleic acid with said first set of primers and said second set of primers under conditions suitable for amplification of said template nucleic acid; and
amplifying said template nucleic acid to produce said library of mutated nucleic acids, wherein said mutated nucleic acids have at least one targeted mutation therein.
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Abstract
The present invention relates generally to in vitro methods for generating mutant nucleic acid and polypeptide libraries. In particular, the present invention provides methods and compositions for mutagenesis and recombination of polynucleotide sequences in amplification methods that utilize primer oligonucleotides, and for expressing the resultant nucleic acid libraries to generate polypeptide libraries.
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Citations
25 Claims
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1. A method of producing a library of mutated nucleic acids, said method comprising:
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providing a single-stranded template nucleic acid comprising a first sequence of nucleotides; providing a first set of primers, wherein a majority of said first set of primers are perfectly complementary to at least a portion of said first sequence of nucleotides; providing a second set of primers, wherein a majority of said second set of primers are complementary to at least a portion of said first sequence of nucleotides except for at least one pre-selected mismatched nucleotide complementary to at least one targeted mutation; combining said template nucleic acid with said first set of primers and said second set of primers under conditions suitable for amplification of said template nucleic acid; and amplifying said template nucleic acid to produce said library of mutated nucleic acids, wherein said mutated nucleic acids have at least one targeted mutation therein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of generating a library of polynucleotide molecules, wherein said polynucleotide molecules encode at least a portion of a mutant enzyme, said method comprising:
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contacting at least one single-stranded template polynucleotide with a set of primers, wherein said set of primers comprises; at least one primer perfectly complementary to at least a portion of said template polynucleotide; and a plurality of mutant primers, wherein each of said plurality of mutant primers comprises at least one pre-selected mismatched nucleotide complementary to at least one targeted mutation; conducting a multi-cycle polynucleotide extension reaction with said at least one template polynucleotide and said set of primers, wherein; in at least one cycle, said primers anneal to said at least one template polynucleotide and prime replication of said at least one template polynucleotide, thereby generating a pool comprising overlapping fragments, wherein said overlapping fragments are shorter in length than said at least one template polynucleotide and wherein said overlapping fragments overlap to span said at least one template polynucleotide molecule; in at least one subsequent cycle, said overlapping fragments generated in a previous cycle anneal in new combinations to said at least one template polynucleotide molecule, thereby forming annealed fragments, wherein said annealed fragments prime replication of said at least one template polynucleotide molecule to form a further pool of overlapping fragments; and wherein said multi-cycle polynucleotide extension reaction is continued for a sufficient number of cycles such that said further pool of overlapping fragments includes variant forms of said at least one template polynucleotide molecule, thereby generating said library of polynucleotide molecules. - View Dependent Claims (21, 22, 23, 24, 25)
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Specification