Multi-site mutagenesis
First Claim
1. An isolated composition comprising a DNA polymerase, a thermostable DNA ligase, a mutagenic primer that is specific for a target polynucleotide, the target polynucleotide, and a flap endonuclease.
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Accused Products
Abstract
The present invention provides compositions and improved methods for multi-site directed mutagenesis and DNA shuffling. The present compositions and methods provide increased mutation frequency and increased number of transformants which allow one to sequence only a few clones in order to identify the correct mutants and to obtain the desired mutant by screening large number of transformants in a short time. Moreover, the inclusion of FEN-1, PEF and optimized buffer and cycling conditions provided in the present invention should also facilitate random mutagenized library construction and the mutagenesis of large or difficult templates.
87 Citations
61 Claims
- 1. An isolated composition comprising a DNA polymerase, a thermostable DNA ligase, a mutagenic primer that is specific for a target polynucleotide, the target polynucleotide, and a flap endonuclease.
- 16. An isolated composition comprising Pfu DNA polymerase, a thermostable DNA ligase, a target nucleic acid for mutagenesis, and flap endonuclease.
- 24. A kit comprising a DNA polymerase, a thermostable DNA ligase, a flap endonuclease, a restriction endonuclease, a mutagenic primer that is specific for a target polynucleotide, a control template nucleic acid, and packaging means therefor.
- 30. A kit comprising Pfu DNA polymerase, Taq DNA ligase, a mutagenic primer that is specific for a target polynucleotide, a control template nucleic acid, and FEN-1.
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33. A kit comprising 2.5 U Pfu DNA polymerase, 15 U Taq DNA ligase, 400 ng flap endonuclease, and 0.1 mM nicotinamide adenine dinucleotide per 25 μ
- l reaction.
- View Dependent Claims (34, 35, 36)
- 37. An isolated composition comprising a DNA polymerase, a thermostable DNA ligase, a restriction endonuclease, a target nucleic acid for mutagenesis, and a flap endonuclease.
- 43. An isolated composition comprising a DNA polymerase, a thermostable DNA ligase, a selection enzyme, a parental target DNA, and a flap endonuclease, wherein said selection enzyme selectively digests the parental target DNA.
- 55. An isolated composition comprising a DNA polymerase, a thermostable DNA ligase, RecJ exonuclease, a target polynucleotide, and a mutagenic primer that is specific for the target polynucleotide.
Specification