Targeted mutagenesis in living cells using modified oligonucleotides
First Claim
1. A modified oligonucleotide for site-directed mutagenesis of a double-stranded nucleic acid molecule comprising one or more mutagens incorporated into a single-stranded oligonucleotide, said oligonucleotide having a sequence that forms a triple-stranded nucleic acid molecule with a target sequence in the double-stranded nucleic acid molecule.
1 Assignment
0 Petitions
Accused Products
Abstract
A method for introducing a site-specific mutation into a target polynucleotide sequence is presented. The method involves the use of an oligonucleotide capable of binding to the target sequence, either by triplex formation (mediated by Hoogsteen, reverse Hoogsteen or equivalent base pairing) or by Watson/Crick base pairing (in the presence of a recombinase enzyme). The oligonucleotide of the invention is modified by the covalent attachment of one or more electrophilic groups. When a modified oligonucleotide is bound to its target sequence, the electrophilic group is able to interact with a nearby nucleotide in the target sequence, causing a modification to the nucleotide that results in a change in nucleotide sequence. Compositions used in the practice of the method are also disclosed.
-
Citations
22 Claims
- 1. A modified oligonucleotide for site-directed mutagenesis of a double-stranded nucleic acid molecule comprising one or more mutagens incorporated into a single-stranded oligonucleotide, said oligonucleotide having a sequence that forms a triple-stranded nucleic acid molecule with a target sequence in the double-stranded nucleic acid molecule.
-
7. A method for site-directed mutagenesis of a nucleic acid molecule comprising the steps of:
-
a) contacting a modified oligonucleotide to a target sequence of a double-stranded nucleic acid molecule, wherein the modified oligonucleotide comprises one or more mutagens incorporated into a single-stranded nucleic acid that forms a triple-stranded nucleic acid molecule with the target region; and b) thereby mutating the double-stranded nucleic acid molecule. - View Dependent Claims (8, 9, 10, 11, 12, 13, 22)
-
-
14. A method of producing a mutagenic oligonucleotide comprising the steps of:
-
a) synthesizing an oligonucleotide which forms a triple-stranded nucleic acid molecule with a target sequence of a double-stranded nucleic acid molecule; and b) covalently attaching one or more mutagens to the oligonucleotide. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21)
-
Specification