Mismatch endonucleases and methods of use
AssigneeLarge Scale Biology Corporation
InventorsHal S. Padgett, Andrew A. Vaewhongs, Fakhrieh S. Vojdani, Mark L. Smith, John A. Lindbo, Wayne P. Fitzmaurice
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Abstract
We describe here restriction endonucleases and their uses. Restriction endonucleases are useful in finding single nucleotide polymorphisms. They are also useful in an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences.
Claims12
An in vitro method of obtaining a peptide sequence a desired functional property, comprising:
- a) preparing at least one heteroduplex polynucleotide sequence wherein said heteroduplex polynucleotide sequence has at least two non-complementary nucleotide base pairs;
- b) mixing copies of the heteroduplex polynucleotide sequence with an effective amount of a mismatch endonuclease or a combination of mismatch endonucleases, a proofreading enzyme, and a ligase enzyme;
- c) allowing sufficient time for non-complementary nucleotide base pairs to be converted to complementary base pairs, wherein a population of polynucleotide sequence variants result.;
- d) expressing polynucleotide sequence variants;
- and
- e) screening or selecting variants for the desired functional property.
A method making a mismatch endonuclease enzyme comprising:
- a) transfecting a host plant, or host animal, or host yeast or host fungus or host bacterium with a recombinant viral vector that encodes a polynucleotide sequence for a mismatch endonuclease;
- b) growing the host plant, or host animal, or host yeast or host fungus or host bacterium wherein the polynucleotide sequence for a mismatch endonuclease enzyme is expressed;
- and
- c) extracting the mismatch endonuclease enzyme from the host.
A method for determining a mutation in a target sequence of a single stranded polynucleotide with reference to a non-mutated sequence of a polynucleotide that is hybridizable with the polynucleotide including said target sequence, wherein said polynucleotides are amplified, labeled with a detectable marker, hybridized to one another, subjected to the activity of an endonuclease and analyzed for the presence of said mutation, the improvement comprising the use of a non-naturally occurring mismatch endonuclease enzyme, or a mismatch endonuclease enzyme of Selaginella lepidophylla origin, the activity of said enzyme comprising:
- a. detection of mismatches whether known or unknown between said hybridized polynucleotides;
- b. catalytic formation of a substantially single-stranded nick at a target sequence containing a mismatch;
- and
- c. recognition of a mutation in a target polynucleotide sequence, said recognition being substantially unaffected by flanking polynucleotide sequences.
An in vitro method of obtaining a polynucleotide sequence encoding a desired functional property, comprising:
- a) preparing at least one heteroduplex polynucleotide sequence wherein said heteroduplex polynucleotide sequence has at least two non-complementary nucleotide base pairs;
- b) mixing copies of the heteroduplex polynucleotide sequence with an effective amount of a mismatch endonuclease or a combination of mismatch endonucleases, a proofreading enzyme, and a ligase enzyme;
- c) allowing sufficient time for non-complementary nucleotide base pairs to be converted to complementary base pairs, wherein a population of polynucleotide sequence variants result;
- and
- d) screening or selecting variants for the desired functional property.
Dependencies are inferred from the claim text.