Programmed sequential mutagenesis
First Claim
1. A method for performing programmed sequential mutagenesis of nucleic acids comprising providing an original template nucleic acid or nucleic acid analog including a first target sequence;
- providing a first mutagenic primer nucleic acid or nucleic acid analog including a first target-binding sequence capable of hybridizing to said first target sequence under conditions permissive for polymerase mediated nucleic acid synthesis, but which is non-complementary to said first target sequence at one or more nucleotide or nucleotide analog positions;
subjecting said original template and said first mutagenic primer to a first round of nucleic acid synthesis to produce a first mutagenized nucleic acid or nucleic acid analog including a second target sequence which is neither identical nor complementary to any sequence in said original template nucleic acid or nucleic acid analog;
providing a second mutagenic primer nucleic acid or nucleic acid analog including a second target-binding sequence capable of hybridizing to said second target sequence under conditions permissive for polymerase mediated nucleic acid synthesis, but which is non-complementary to said second target sequence atone or more nucleotide or nucleotide analog positions; and
subjecting said first mutagenized nucleic acid or nucleic acid analog and said second mutagenic primer to a second round of nucleic acid synthesis to produce a second mutagenized nucleic acid or nucleic acid analog including a third target sequence which is neither identical nor complementary to any sequence in said original template or first mutagenized nucleic acid or nucleic acid analogs.
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Accused Products
Abstract
Methods and products are provided for the oligonucleotide-mediated polymerase-dependent mutation of template nucleic acid sequences in a programmed sequence over multiple rounds of nucleic acid synthesis. The methods employ series of mutagenic primers, all of which may be present in the initial reaction system, in which at least some of the mutagenic primers do not bind to or promote polymerization from the original template nucleic acids, but do bind to and promote polymerization from the mutagenized products of earlier rounds of the program of sequential mutagenesis. The products of the invention include mixtures of such mutagenic primers. The products and methods have applications in the biological and computational sciences.
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Citations
13 Claims
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1. A method for performing programmed sequential mutagenesis of nucleic acids comprising providing an original template nucleic acid or nucleic acid analog including a first target sequence;
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providing a first mutagenic primer nucleic acid or nucleic acid analog including a first target-binding sequence capable of hybridizing to said first target sequence under conditions permissive for polymerase mediated nucleic acid synthesis, but which is non-complementary to said first target sequence at one or more nucleotide or nucleotide analog positions;
subjecting said original template and said first mutagenic primer to a first round of nucleic acid synthesis to produce a first mutagenized nucleic acid or nucleic acid analog including a second target sequence which is neither identical nor complementary to any sequence in said original template nucleic acid or nucleic acid analog;
providing a second mutagenic primer nucleic acid or nucleic acid analog including a second target-binding sequence capable of hybridizing to said second target sequence under conditions permissive for polymerase mediated nucleic acid synthesis, but which is non-complementary to said second target sequence atone or more nucleotide or nucleotide analog positions; and
subjecting said first mutagenized nucleic acid or nucleic acid analog and said second mutagenic primer to a second round of nucleic acid synthesis to produce a second mutagenized nucleic acid or nucleic acid analog including a third target sequence which is neither identical nor complementary to any sequence in said original template or first mutagenized nucleic acid or nucleic acid analogs. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
providing a first outer primer including a sequence capable of hybridizing to said template nucleic acid at a site 3′ - of said first target sequence under conditions permissive for polymerase mediated nucleic acid synthesis.
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7. A method as in any one of claims 1-3 further comprising
providing a second outer primer including a sequence capable of hybridizing to said first mutagenized nucleic acid at a site 3′ - of said second target sequence under conditions permissive for polymerase mediated nucleic acid synthesis.
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8. A method as in any one of claims 1-3 wherein said target sequences comprise a sequence of at least 10 nucleotides or nucleotide analogs.
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9. A method as in any one of claims 1-3 wherein said target sequences comprise a sequence of at least 15 nucleotides or nucleotide analogs.
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10. A method as in any one of claims 1-3 wherein said target sequences comprise a sequence of at least 20 nucleotides or nucleotide analogs.
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11. A method as in claim 8 wherein said mutagenic primers comprise a sequence of at least 10 nucleotides or nucleotide analogs;
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wherein said target-binding sequences are non-complementary to their target sequences at 1-4 nucleotides or nucleotide analog positions.
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12. A method as in claim 9 wherein said mutagenic primers comprise a sequence of at least 15 nucleotides or nucleotide analogs;
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wherein said target-binding sequences are non-complementary to their target sequences at 1-7 nucleotides or nucleotide analog positions.
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13. A programmed sequential mutagenesis reagent comprising
a first set of one or more mutagenic primers which will bind to one or more target sequences within a template sequence under first conditions permissive for polymerase-mediated nucleic acid synthesis to create a first set of one or more mutagenized products; - and
a second set of one or more mutagenic primers which will not bind to any target sequence within said template sequence under said first conditions, but which will bind to one or more target sequences in said first set of mutagenized products under conditions permissive for polymerase-mediated nucleic acid synthesis to create a second set of one or more mutagenized products.
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Specification