Method of generating targeted dna libraries
AssigneeTrustees of Tufts College
InventorsAlex A. Bohm, Gretchen Meinke, Nahide Dalda
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Abstract
The present invention provides methods and kits for efficiently generating edited DNA, ranging from DNA molecules with simple point mutations to complex DNA libraries. The methods and kits use a single-stranded deoxyuridine (dU)-containing template to align a series of DNA oligomers that contain mutations relative to the template strand, giving rise to the sequence variation in the product. The methods and kits may be used to produce DNA libraries or to generate multiple, widely spaced mutations in a target sequence.
Claims20
A method of generating edited DNA comprising:
- a. amplifying a template via polymerase chain reaction (PCR) using a first phosphorylated primer, a second nonphosphorylated primer, and a DNA polymerase capable of incorporating deoxyuridine triphosphate (dUTP) to generate a deoxyuridine (dU)-containing amplicon;
- b. digesting the dU-containing amplicon of step (a) to degrade the phosphorylated strand and produce a single-stranded, dU-containing template;
- c. annealing at least one mutagenic primer to the template of step (b), wherein the mutagenic primer is 5′ phosphorylated single-stranded DNA comprising an edited region flanked on each side by at least 10 bases that are complementary to the template, and wherein the edited region comprises at least one mutation relative to the complement of the template;
- d. extending the mutagenic primers annealed in step (c) using a DNA polymerase that can read through uracil present in DNA and lacks 3′-exonuclease activity, 5′-exonuclease activity, and strand displacement activity to produce an extension product;
- e. ligating the extension product of step (d) to produce a partially mismatched double-stranded DNA product comprising a ligated edited strand and the dU-containing template;
- f. amplifying the edited strand of step (e) using a forward primer, a reverse primer, and a DNA polymerase.
A kit for generating edited DNA comprising:
- a. a first DNA polymerase that is capable of incorporating dUTP and reading through uracil present in DNA, and that lacks 3′-exonuclease activity, 5′-exonuclease activity, and strand displacement activity;
- b. a second DNA polymerase that is not capable of reading through uracil present in DNA;
- c. at least two deoxynucleotide triphosphate (dNTP) mixes, wherein at least one dNTP mix contains dU and at least one dNTP mix does not contain dU;
- and
- d. a lambda (λ) exonuclease.
Dependencies are inferred from the claim text.