Compositions and methods for the construction of a random allelic series
AssigneesWashington University, Shriners Hospitals for Children
InventorsChristine Gurnett, Gabriel Haller, Matthew Dobbs, David Alvarado
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Abstract
The present disclosure provides a method of making a systematic single point mutation in a target nucleic acid and a method of generating a mutational library comprising target nucleic acids with single point mutations. The mutational library comprises target nucleic acids with single point mutations distributed evenly throughout the target nucleic acid.
Claims20
A method of generating a mutational library of a target nucleic acid, the method comprising:
- a) creating a reaction mixture comprising the target nucleic acid, polymerase, a forward primer, deoxynucleotide triphosphates (dNTPs) and universal base triphosphates comprising a blocking group;
- b) performing linear PCR, wherein the linear PCR generates products of various lengths, wherein each product comprises about 1 universal base comprising a blocking group, and wherein the linear PCR stops after incorporating the universal base triphosphate comprising a blocking group;
- c) removing the blocking group from the universal base incorporated into the linear product;
- d) extending the products from (c) with polymerase, wherein the extension products comprise about 1 universal base incorporated therein;
- and
- e) performing exponential PCR, wherein the products from (d) are amplified and wherein the products comprise a single point mutation in the location where the universal base was incorporated;
- and
- wherein the products of the exponential PCR make up a mutational library of the target nucleic acid comprising a plurality of products each comprising a single point mutation, wherein the point mutations are evenly distributed throughout the products of the mutational library.
A method of making 2′-deoxy, 3′-O—NH2 inosine triphosphate from 2′-deoxy, 3′-O—NH2 adenosine triphosphate, the method comprising:
- a) dephosphorylating 2′-deoxy, 3′-O—NH2 adenosine triphosphate with a phosphatase;
- b) deaminating the product from (a) with a deaminase;
- and
- c) phosphorylating the product from (b) with three different kinases.
Dependencies are inferred from the claim text.