Method of increasing complementarity in a heteroduplex
First Claim
1. An in vitro method of making sequence variants from at least one heteroduplex polynucleotide where said heteroduplex has at least two non-complementary nucleotide base pairs, said method comprising:
- a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of CEL I, T4 DNA polymerase, and T4 DNA ligase; and
c. allowing sufficient time for the percentage of complementarity to increase, wherein one or more variants are made.
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Abstract
We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.
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Citations
65 Claims
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1. An in vitro method of making sequence variants from at least one heteroduplex polynucleotide where said heteroduplex has at least two non-complementary nucleotide base pairs, said method comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of CEL I, T4 DNA polymerase, and T4 DNA ligase; and
c. allowing sufficient time for the percentage of complementarity to increase, wherein one or more variants are made.
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2. An in vitro method of making sequence variants from at least one heteroduplex polynucleotide wherein said heteroduplex has at least two non-complementary nucleotide base pairs, said method comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of an agent or agents with exonuclease activity, polymerase activity and strand cleavage activity; and
c. allowing sufficient time for the percentage of complementarity to increase, wherein at least one or more variants are made. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 61, 62, 63)
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27. An in vitro method of increasing diversity in a population of sequences, comprising:
- preparing at least one heteroduplex polynucleotide;
combining said heteroduplex polynucleotide with an effective amount of an agent or agents with 3′
to 5′
exonuclease activity, polymerase activity and strand cleavage activity; and
allowing sufficient time for the percentage of complementarity to increase, wherein diversity in the population is increased.
- preparing at least one heteroduplex polynucleotide;
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50. An in vitro method of increasing diversity in a population of sequences, comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of CEL I, T4 DNA polymerase, and T4 DNA ligase; and
c. allowing sufficient time for the percentage of complementarity to increase, wherein diversity in the population is increased.
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51. A method of obtaining a polynucleotide encoding a desired functional property, comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of an agent or agents with exonuclease activity, polymerase activity, and strand cleavage activity;
c. allowing sufficient time for the percentage of complementarity between strands of the heteroduplex polynucleotide to increase, wherein diversity in the population is increased; and
d. screening or selecting a population of variants for the desired functional property.
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52. A method of obtaining a polynucleotide encoding a desired functional property, comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of an agent or agents with exonuclease activity, polymerase activity, and strand cleavage activity;
c. allowing sufficient time for the percentage of complementarity between strands of the heteroduplex polynucleotide to increase, wherein diversity in the population is increased;
d. converting DNA to RNA; and
e. screening or selecting a population of ribonucleic acid variants for the desired functional property.
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53. A method of obtaining a polypeptide having a desired functional property, comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of an agent or agents with exonuclease activity, polymerase activity and strand cleavage activity;
c. allowing sufficient time for the percentage of complementarity between strands of said heteroduplex polynucleotide to increase, d. converting said heteroduplex polynucleotide to RNA, and said RNA to a polypeptide; and
e. and screening or selecting a population of polypeptide variants for said desired functional property.
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54. A method of obtaining a polynucleotide encoding a desired functional property, comprising:
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a. preparing at least one heteroduplex polynucleotide;
b. combining said heteroduplex polynucleotide with an effective amount of an agent or agents with exonuclease activity, polymerase activity and strand cleavage activity;
c. allowing sufficient time for the percentage of complementarity between strands of said heteroduplex polynucleotide to increase, d. screening or selecting for a population of variants having a desired functional property;
e. denaturing said population of variants to obtain single strand polynucleotides;
f. annealing said single strand polynucleotides to form at least one second heteroduplex polynucleotide;
g. combining said second heteroduplex polynucleotide with an effective amount of an agent or agents with exonuclease activity, polymerase activity and strand cleavage activity; and
h. allowing sufficient time for the percentage of complementarity between strands of the heteroduplex polynucleotide to increase. - View Dependent Claims (55, 56, 57, 58, 59, 60)
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64. A kit used for increasing diversity in a population of sequences, comprising:
- preparing at least one heteroduplex polynucleotide;
combining said heteroduplex polynucleotide with an effective amount of an agent or agents with 3′
to 5′
exonuclease activity, polymerase activity and strand cleavage activity; and
allowing sufficient time for the percentage of complementarity to increase, wherein diversity in the population is increased. - View Dependent Claims (65)
- preparing at least one heteroduplex polynucleotide;
Specification