US6117679A
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US
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Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
Assignee: Codexis Inc
•
Filing: 1996-03-25
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Granted: 2000-09-12
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Expires: 2016-03-25
Abstract
Abstract A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.
Inventors
- 👤 Willem P. C. Stemmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 04:15
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K14/00; C07K14/435; C07K14/43504; C07K14/43595; C07K14/52; C07K14/54; C07K14/545; C07K16/00; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N15/1027; C12N15/1034; C12N15/1037; C12N15/1058; C12N15/11; C12N15/52; C12N15/63; C12N15/64; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; C12N9/78; C12N9/86; C12Q; C12Q1/00; C12Q1/68; C12Q1/6811; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; C12Y305/00; C12Y305/02; C12Y305/02006; H; H10; H10N; H10N60/00; H10N60/10; H10N60/128; C07K2317/00; C07K2317/50; C07K2317/56; C07K2317/565; C07K2317/60; C07K2317/62; C07K2317/622
Claims Analysis
Total Claims: 55
Claim 1
Independent Claim
71 words
A method of shuffling polynucleotides, comprising: conducting a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides; and selecting or screening a recombinant polynucleotide for a desired property, wherein the amplification process is performed in the presence of an agent that promotes annealing of the overlapping segments.
Claim 2
Independent Claim
71 words
A method of shuffling polynucleotides, comprising: conducting a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides; and selecting or screening a recombinant polynucleotide for a desired property, wherein the amplification process is performed in the presence of an agent that promotes annealing of the overlapping segments.
Claim 3
Dependent Claim
24 words
The method of claim 1, wherein the agent is selected from the group consisting of, a cationic detergent, an exonuclease and a recombinogenic protein.
Claim 4
Dependent Claim
10 words
The method of claim 1, wherein the agent is recA.
Claim 5
Independent Claim
87 words
A method of shuffling polynucleotides, comprising: conducting a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides; and selecting or screening a recombinant polynucleotide for a desired property wherein the population of variant polynucleotides is converted into overlapping segments of a desired size by replication of the polynucleotide in the presence of UTP, cleavage of the replicated polynucleotide with UDG glycosylase and denaturation.
Claim 6
Independent Claim
87 words
A method of shuffling polynucleotides, comprising: conducting a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides; and selecting or screening a recombinant polynucleotide for a desired property wherein the population of variant polynucleotides is converted into overlapping segments of a desired size by replication of the polynucleotide in the presence of UTP, cleavage of the replicated polynucleotide with UDG glycosylase and denaturation.
Claim 7
Independent Claim
147 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is between different forms of the polynucleotide sequence in separate plasmid vectors.
Claim 8
Independent Claim
147 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is between different forms of the polynucleotide sequence in separate plasmid vectors.
Claim 9
Independent Claim
160 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property wherein at least one recombining step is between a first form of the polynucleotide sequence in a viral vector and a second form of the polynucleotide sequence in a plasmid vector.
Claim 10
Independent Claim
160 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property wherein at least one recombining step is between a first form of the polynucleotide sequence in a viral vector and a second form of the polynucleotide sequence in a plasmid vector.
Claim 11
Independent Claim
147 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is between different forms of the polynucleotide sequences in separate viral vectors.
Claim 12
Independent Claim
147 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is between different forms of the polynucleotide sequences in separate viral vectors.
Claim 13
Independent Claim
161 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is between a first form of the polynucleotide and a second form of the polynucleotide that is a component of a chromosome in a host cell.
Claim 14
Independent Claim
161 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is between a first form of the polynucleotide and a second form of the polynucleotide that is a component of a chromosome in a host cell.
Claim 15
Independent Claim
141 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the first form of the polynucleotide is in a plasmid vector.
Claim 16
Independent Claim
141 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the first form of the polynucleotide is in a plasmid vector.
Claim 17
Independent Claim
141 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the first form of the polynucleotide is in a viral vector.
Claim 18
Independent Claim
141 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the first form of the polynucleotide is in a viral vector.
Claim 19
Independent Claim
160 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein different forms of the polynucleotide are contained in a population of cells and the cells are exposed to an electric field promoting exchange of the different forms between the cells.
Claim 20
Independent Claim
160 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein different forms of the polynucleotide are contained in a population of cells and the cells are exposed to an electric field promoting exchange of the different forms between the cells.
Claim 21
Independent Claim
152 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein different forms of the polynucleotide are contained in a collection of cells and the different forms are exchanged between cells by conjugation.
Claim 22
Independent Claim
152 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein different forms of the polynucleotide are contained in a collection of cells and the different forms are exchanged between cells by conjugation.
Claim 23
Independent Claim
146 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is effected by nonhomologous recombination of different forms of the polynucleotide.
Claim 24
Independent Claim
146 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is effected by nonhomologous recombination of different forms of the polynucleotide.
Claim 25
Independent Claim
155 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the polynucleotide has introns and exons and at least one recombining step is effected by homologous recombination between introns of different forms of the polynucleotide.
Claim 26
Independent Claim
155 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the polynucleotide has introns and exons and at least one recombining step is effected by homologous recombination between introns of different forms of the polynucleotide.
Claim 27
Independent Claim
149 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is performed in vivo and at least one recombining step is performed in vitro.
Claim 28
Independent Claim
149 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is performed in vivo and at least one recombining step is performed in vitro.
Claim 29
Independent Claim
139 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least two recombining steps are performed in vitro.
Claim 30
Independent Claim
139 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least two recombining steps are performed in vitro.
Claim 31
Independent Claim
152 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property and (6) selecting for recombinant or further recombinant polynucleotides relative to unrecombined forms of the polynucleotide sequence after at least one recombining step.
Claim 32
Independent Claim
152 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property and (6) selecting for recombinant or further recombinant polynucleotides relative to unrecombined forms of the polynucleotide sequence after at least one recombining step.
Claim 33
Independent Claim
150 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is performed in a mutator host cell or a host cell exposed to a mutagen.
Claim 34
Independent Claim
150 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein at least one recombining step is performed in a mutator host cell or a host cell exposed to a mutagen.
Claim 35
Independent Claim
136 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the polynucleotide sequence is a gene.
Claim 36
Independent Claim
136 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the polynucleotide sequence is a gene.
Claim 37
Dependent Claim
17 words
The method of claim 19, wherein at least one form of the polynucleotide is in purified form.
Claim 38
Dependent Claim
15 words
The method of claim 19, wherein at least one recombining step is performed in vivo.
Claim 39
Dependent Claim
16 words
(Amended) The method of claim 19, wherein at least two recombining steps are performed in vivo.
Claim 40
Independent Claim
149 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the further recombinant polynucleotide evolves at a frequency of at least 1 mutation per 10 6 positions per cycle.
Claim 41
Independent Claim
149 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the further recombinant polynucleotide evolves at a frequency of at least 1 mutation per 10 6 positions per cycle.
Claim 42
Independent Claim
147 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the second form of the polynucleotide is produced by mutagenesis of the first form of the polynucleotide.
Claim 43
Independent Claim
147 words
A method of evolving a polynucleotide sequence toward a desired property comprising: (1) recombining at least first and second forms of the polynucleotide sequence to produce a library of recombinant forms of the sequence; (2) screening at least a first recombinant form of the polynucleotide from the library for evolution toward the desired property; (3) recombining the first recombinant form of the polynucleotide with a further form of the polynucleotide sequence, the same or different from the first and second forms, to produce a further library of recombinant polynucleotides; (4) screening at least one further recombinant polynucleotide from the further library of recombinant polynucleotides that has further evolved toward the desired property; (5) repeating (3) and (4), as necessary, until the further recombinant polynucleotide has acquired the desired property, wherein the second form of the polynucleotide is produced by mutagenesis of the first form of the polynucleotide.
Claim 44
Dependent Claim
49 words
The method of claim 16, wherein the in vitro recombining steps comprise: conducting a polynucleotide amplification process on overlapping single-stranded segments of a population of variant polynucleotidcs under conditions whereby one segment serves a s a template for extension of another segment to generate a population of recomnbinant polynucleotides.
Claim 45
Dependent Claim
47 words
The method of claim 19, wherein at least one recombining step comprises: conducting a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides.
Claim 46
Independent Claim
52 words
A method of shuffling polynucleotides, comprising: initiating a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynuclcotides: and selecting or screening a recombinant polynucleotide for a desired property.
Claim 47
Independent Claim
52 words
A method of shuffling polynucleotides, comprising: initiating a polynucleotide amplification process on overlapping segments of a population of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynuclcotides: and selecting or screening a recombinant polynucleotide for a desired property.
Claim 48
Independent Claim
25 words
The method of any one of claims 25, 26 and 27, wherein the overlapping segments are produced by cleavage of the population of variant polynucleotides.
Claim 49
Independent Claim
18 words
The method of any one of claims 25, 26, and 27, wherein the cleavage is by DNaseI digestion.
Claim 50
Independent Claim
20 words
The method of any one of claims 25, 26 and 27, wherein the overlapping segments are produced by chemical synthesis.
Claim 51
Independent Claim
24 words
The method of any one of claims 25, 26 and 27, wherein the overlapping segments are produced by amplification of the population of polynucleotides.
Claim 52
Independent Claim
20 words
The method of any one of claims 25, 26 and 27, wherein the population of variant polynucleotides are allelic variants.
Claim 53
Independent Claim
20 words
The method of any one of claims 25, 26 and 27, wherein the population of variant polynucleotides are species variants.
Claim 54
Dependent Claim
38 words
The method of claim 25, wherein the overlapping single-stranded segments are produced by: cleaving the population of variant polynucleotides to produce overlapping double-stranded polynucleotide fragments; and denaturing the double-stranded polynucleotide fragments to produce the overlapping single-stranded polynucleotide segments.
Claim 55
Dependent Claim
19 words
The method of claim 34, wherein the variant polynucleotides are DNA and the cleaving is performed by DNase digestion.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-09-12 | 2004-03-13 | 2004-09-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-09-12 | 2008-03-13 | 2008-09-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-09-12 | 2012-03-13 | 2012-09-12 | $7700.00 | Window Closed / Lapsed |