Process for conducting site-directed mutagenesis
First Claim
1. A process for conducting site-directed mutagenesis on double-stranded DNA previously cleaved and denatured to single-stranded linear DNA templates, said process comprising(a) hybridizing mutagenic and closing oligonucleotides to single-stranded linear DNA templates, said closing oligonucleotide being characterized by having a nucleotide sequence complementary to one or both of the free ends of said linear DNA templates, and functioning to circularize said linear DNA templates and generate a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently-closed heteroduplex DNA molecule,(b) incorporating said mutagenic and closing oligonucleotides into DNA strands complementary to said linear DNA templates by submitting said hybridized linear DNA templates to the action of polymerase and ligase enzymes to yield heteroduplex double-stranded DNA molecules, and(c) selectively replicating said complementary DNA strands of said heteroduplex double-stranded DNA.
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Abstract
The present invention relates to an approach for conducting site-directed mutagenesis using double-stranded DNA templates. The approach involves the development of a method for generating structures capable of directing full-length complementary-strand synthesis from double-stranded plasmid DNA. These structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing oligonucleotide". A "closing oligonucleotide" is a single-stranded oligonucleotide consisting of a sequence complementary to either or both free ends of one of the two plasmid DNA strands. The "closing oligonucleotide" therefore functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently-closed heteroduplex DNA molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.
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Citations
11 Claims
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1. A process for conducting site-directed mutagenesis on double-stranded DNA previously cleaved and denatured to single-stranded linear DNA templates, said process comprising
(a) hybridizing mutagenic and closing oligonucleotides to single-stranded linear DNA templates, said closing oligonucleotide being characterized by having a nucleotide sequence complementary to one or both of the free ends of said linear DNA templates, and functioning to circularize said linear DNA templates and generate a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently-closed heteroduplex DNA molecule, (b) incorporating said mutagenic and closing oligonucleotides into DNA strands complementary to said linear DNA templates by submitting said hybridized linear DNA templates to the action of polymerase and ligase enzymes to yield heteroduplex double-stranded DNA molecules, and (c) selectively replicating said complementary DNA strands of said heteroduplex double-stranded DNA.
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8. A kit for conducting site-directed mutagenesis of double-stranded DNA previously cleaved and denatured to single-stranded linear DNA templates, said templates being hybridized to mutagenic and closing oligonucleotides incorporated into DNA strands complementary to said templates, said kit comprising:
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(a) one or more closing oligonucleotides having a nucleotide sequence complementary to one or both of the free ends of said linear DNA templates, said oligonucleotide being characterized and functioning to circularize said linear DNA templates and generate a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently-closed heteroduplex DNA molecule, and (b) means for selective replication of said complementary DNA strands. - View Dependent Claims (9, 10, 11)
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Specification