Population of polynucleotide sequence variants
AssigneeNovici Biotech LLC
InventorsHal S. Padgett, John A. Lindbo, Wayne P. Fitzmaurice
Life of this patent
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Abstract
We describe here an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences, by making a heteroduplex polynucleotide from two non-identical polynucleotides; introducing a nick in one strand at or near a base pair mismatch site; removing mismatched base(s) from the mismatch site where the nick occurred; and using the opposite strand as template to replace the removed base(s) with bases that complement base(s) in the first strand. By this method, information is transferred from one strand to the other at sites of mismatch.
Claims17
An in vitro method of redistributing sequence variations between non-identical polynucleotides wherein each step is performed outside of living cells, comprising:
- a) making a heteroduplex polynucleotide from two non-identical polynucleotides having 96% or more sequence identity, the heteroduplex having first and second strands;
- b) mixing the heteroduplex polynucleotide with an effective amount of mismatch-directed strand cleavage activity, proofreading activity, and ligase activity wherein said mismatch-directed strand cleavage activity consists of a single enzyme that recognizes a mismatch directly;
- and
- c) allowing sufficient time for at least two non-complementary nucleotide base pairs to be converted to complementary base pairs wherein a population of polynucleotide strands not identical to either of said first and second strands results and wherein the polynucleotide strands of the population have an average of 1 or more crossovers per 120 basepairs relative to the distance between the first and last mismatches.
An in vitro method of making a population containing sequence variants from a heteroduplex polynucleotide wherein each step is performed outside of living cells, comprising:
- a) making a heteroduplex polynucleotide from two non-identical polynucleotides having 54% or more sequence identity;
- b) mixing copies of the heteroduplex polynucleotide with an effective amount of a mismatch-directed mismatch endonuclease, a proofreading enzyme, and a ligase enzyme wherein said mismatch-directed mismatch endonuclease consists of a single enzyme that recognizes a mismatch directly;
- and
- c) allowing sufficient time for a number of non-complementary nucleotide base pairs to be converted to complementary base pairs, wherein a population containing polynucleotide sequence variants results and wherein at least 20% of the population has undergone reassortment of sequence information and the sequence variants have an average of 1 or more crossovers per 250 basepairs of heteroduplex polynucleotide sequence.
An in vitro method of obtaining a polynucleotide encoding a desired functional property, comprising:
- a) preparing at least one heteroduplex polynucleotide outside of living cells;
- b) mixing copies of the heteroduplex polynucleotide with an effective amount of a mismatch-directed mismatch endonuclease, a proofreading enzyme, and a ligase enzyme outside of living cells wherein said mismatch-directed mismatch endonuclease consists of a single enzyme that recognizes a mismatch directly;
- c) allowing sufficient time for a number of non-complementary nucleotide base pairs to be converted to complementary base pairs, wherein a population of at least 85% sequence variants results outside of living cells, and wherein the increased sequence diversity in said population of sequence variants is larger than the increased sequence diversity resulting from a control reaction lacking said mismatch endonuclease;
- and
- d) screening or selecting a population of sequence variants for the desired functional property.
A method of identifying a reassorted DNA molecule encoding a protein with a desired functional property, comprising:
- a) providing at least one single-stranded uracil-containing DNA molecule, which single-stranded uracil-containing molecule, or a complementary strand thereto, encodes a protein;
- b) providing one or a plurality of non-identical single-stranded DNA molecules capable of hybridizing to the single-stranded uracil-containing molecule, wherein said DNA molecules encode at least one additional variant of the protein;
- c) contacting the single-stranded uracil-containing molecule with at least one single-stranded DNA molecule of step (b), thereby producing an annealed DNA molecule;
- d) incubating the annealed DNA molecule with a mismatch-directed mismatch endonuclease, proofreading enzyme and a ligase outside of living cells wherein said mismatch-directed mismatch endonuclease consists of a single enzyme that recognizes a mismatch directly, thereby producing a reassorted DNA strand annealed to the uracil-containing DNA molecule;
- e) amplifying the reassorted DNA strand under conditions wherein the uracil-containing DNA molecule is not amplified, thereby producing a population of reassorted DNA molecules wherein at least 44% of the population has undergone reassortment of sequence information;
- and,
- f) screening or selecting the population of reassorted DNA molecules to identify those that encode a polypeptide having the desired functional property, thereby identifying one or more DNA molecules(s) that encode a polypeptide with the desired functional property.
Dependencies are inferred from the claim text.