Patent US2979170A successfully refreshed from Google Patents.
US6132970A
Expired / Lapsed
US
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Methods of shuffling polynucleotides
Assignee: Codexis Inc
•
Filing: 1998-06-19
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Granted: 2000-10-17
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Expires: 2018-06-19
Abstract
Abstract The invention is directed to methods of shuffling polynucleotide variants. The methods entail conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the plurality of chosen polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of shuffled polynucleotides. Shuffled polynucleotides are then selected or screened to identify a shuffled polynucleotide having a desired functional property.
Inventors
- 👤 Willem P. C. Stemmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 10:10
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; 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; C40B40/04; C40B40/06; C40B40/08; C40B50/00; C40B50/06; C07K2317/00; C07K2317/50; C07K2317/56; C07K2317/565; C07K2317/60; C07K2317/62; C07K2317/622
Claims Analysis
Total Claims: 50
Claim 1
Independent Claim
101 words
A method of shuffling polynucleotides, comprising: choosing a plurality of existing polynucleotide variants to be shuffled; conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the plurality of chosen polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of shuffled polynucleotides; selecting or screening shuffled polynucleotides to identify a shuffled polvnucleotide having a desired functional property.
Claim 2
Independent Claim
101 words
A method of shuffling polynucleotides, comprising: choosing a plurality of existing polynucleotide variants to be shuffled; conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the plurality of chosen polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of shuffled polynucleotides; selecting or screening shuffled polynucleotides to identify a shuffled polvnucleotide having a desired functional property.
Claim 3
Independent Claim
69 words
A method of shuffling polynucleotides, comprising: conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of shuffled polynucleotides; and screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property.
Claim 4
Independent Claim
69 words
A method of shuffling polynucleotides, comprising: conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from a plurality of polynucleotide variants under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of shuffled polynucleotides; and screening or selecting the shuffled polynucleotides to identify a shuffled polynucleotide having a desired functional property.
Claim 5
Dependent Claim
47 words
The method of claim 1 or claim 2, wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the polynucleotide variants and incubating under annealing conditions whereby the single stranded segments from different polynucleotide variants anneal to form the partially annealed polynucleotide strands.
Claim 6
Dependent Claim
17 words
The method of claim 3, wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.
Claim 7
Dependent Claim
17 words
The method of claim 3, wherein the overlapping single-stranded segments are nonrandom segments of the polynucleotide variants.
Claim 8
Dependent Claim
40 words
The method of claim 3, wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments; and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.
Claim 9
Dependent Claim
21 words
The method of claim 6, wherein the population of polynucleotide variants are DNA and the cleavage is by DNase I digestion.
Claim 10
Dependent Claim
17 words
The method of claim 1 or claim 2, further comprising amplifying the chosen plurality of polynucleotide variants.
Claim 11
Dependent Claim
22 words
The method of claim 1 or claim 2, further comprising performing error-prone PCR on at least one of the chosen polynucleotide variants.
Claim 12
Dependent Claim
20 words
The method of claim 1 or claim 2, wherein the partially annealed polynucleotide strands are produced on a DNA synthesizer.
Claim 13
Dependent Claim
24 words
The method of claim 1, or claim 2 wherein the plurality of polynucleotide variants to be shuffled are naturally occurring variants of a polynucleotide.
Claim 14
Dependent Claim
16 words
The method of claim 11, wherein the naturally occurring variants of the polynucleotide encode natural polypeptides.
Claim 15
Dependent Claim
17 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise a human polynucleotide.
Claim 16
Dependent Claim
17 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise a mouse polynucleotide.
Claim 17
Dependent Claim
21 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise a human polynucleotide and a mouse polynucleotide.
Claim 18
Dependent Claim
16 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise bacterial polynucleotides.
Claim 19
Dependent Claim
16 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise plant polynucleotides.
Claim 20
Dependent Claim
16 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise fungal polynucleotidcs.
Claim 21
Dependent Claim
16 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise animal polynucleotides.
Claim 22
Dependent Claim
16 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise viral polynucleotides.
Claim 23
Dependent Claim
32 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprise nonallelic gene sequences from the same species having sufficient sequence similarity for annealing of segments of different variants.
Claim 24
Dependent Claim
19 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotides comprise allelic variants of a gene.
Claim 25
Dependent Claim
19 words
The method of claim 11, wherein the naturally occurring variants of a polynucleotide comprises species variants of a gene.
Claim 26
Dependent Claim
16 words
The method of claim 11, wherein the desired functional property is capacity to bind a receptor.
Claim 27
Dependent Claim
13 words
The method of claim 11, wherein the desired functional property is drug resistance.
Claim 28
Dependent Claim
14 words
The method of claim 11, wherein the desired functional property is a therapeutic activity.
Claim 29
Dependent Claim
18 words
The method of claim 11, wherein the desired functional property is suitability as an agent for gene therapy.
Claim 30
Dependent Claim
18 words
The method of claim 11, wherein the desired functional property is suitability as an agent for anti-neoplastic therapy.
Claim 31
Dependent Claim
18 words
The method of claim 11, wherein the desired functional property is suitability as an agent for DNA-based vaccination.
Claim 32
Dependent Claim
44 words
The method of claim 11, further comprising shuffling a recombinant polynucleotide with the desired property with a naturally occurring polynucleotide to eliminate variations between the recombinant polynucleotide and the naturally occurring polynucleotide that do not contribute to the desired property of the recombinant polynucleotide.
Claim 33
Dependent Claim
26 words
The method of claim 11, further comprising supplementing the plurality of natural variants of a polynucleotide with induced variants of a polynucleotide before the amplification process.
Claim 34
Dependent Claim
22 words
The method of claim 11, further comprising randomly fragmenting the population of natural variants of a polynucleotide before conducting the amplification process.
Claim 35
Dependent Claim
21 words
The method of claim 1 or claim 2, wherein the polynucleotide extension process is conducted on at least 100 polynucleotide strands.
Claim 36
Dependent Claim
28 words
The method of claim 1 claim 2, wherein the concentration of one specific nucleic acid strand is less than 1% by weight of the total number of strands.
Claim 37
Dependent Claim
21 words
The method of claim 1 or claim 2, wherein the polynucleotide strands have sequences from random segments of the polynucleotide variants.
Claim 38
Dependent Claim
19 words
The method of claim 1 or claim 2, wherein the conducting and selecting or screening steps are performed iteratively.
Claim 39
Dependent Claim
33 words
The method of claim 1 or claim 2, wherein at least one variant polynucleotide shows at least 80% sequence identity to a second variant polynucleotide throughout the sequence of the second variant polynucleotide.
Claim 40
Dependent Claim
24 words
The method of claim 1 or claim 2, further comprising expressing the shuffled polynucleotide having the desired function property to produce a shuffled protein.
Claim 41
Dependent Claim
12 words
The method of claim 38, wherein the shuffled protein is an enzyme.
Claim 42
Dependent Claim
13 words
The method of claim 38, wherein the shuffled protein is an antibody chain.
Claim 43
Independent Claim
14 words
The method of cliam 38, wherein the shuffled protein is a viral coat protein.
Claim 44
Independent Claim
14 words
The method of cliam 38, wherein the shuffled protein is a viral coat protein.
Claim 45
Dependent Claim
27 words
The method of claim 1 or 2, wherein the selecting or screening step comprises introducing the shuffled polynucleotides into cells and detecting a property of the cells.
Claim 46
Dependent Claim
35 words
The method of claim 1 or 2, wherein the plurality of polynucleotide variants comprises at least two variants having a plurality of regions of similarity and a plurality of regions of heterology to each other.
Claim 47
Dependent Claim
21 words
The method of claim 1, wherein the desired functional property is an enhanced biological activity relative to an existing polynucleotide variant.
Claim 48
Dependent Claim
17 words
The method of claim 1, wherein the existing polynucleotide variants have a length greater than 50 bases.
Claim 49
Dependent Claim
12 words
The method of claim 1, wherein the heterlogous regions are point mutations.
Claim 50
Dependent Claim
17 words
The method of claim 2, wherein the plurality of polynucleotide variants differ from each by point mutations.
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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) | 2003-10-17 | 2004-04-17 | 2004-10-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-10-17 | 2008-04-17 | 2008-10-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-10-17 | 2012-04-17 | 2012-10-17 | $7700.00 | Window Closed / Lapsed |