Patent US6159690A successfully refreshed from Google Patents.
US6159690A
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Method for in vitro molecular evolution of protein function using exonuclease and amplification
Assignee: Alligator Bioscience AB
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Filing: 1998-06-16
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Granted: 2000-12-12
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Expires: 2018-06-16
Abstract
Abstract The present invention relates to a method for in vitro evolution of protein function. In particular, the method relates to the shuffling of nucleotide segments obtained from exonuclease digestion. The present inventors have shown that polynucleotide fragments derived from a parent polynucleotide sequence digested with an exonuclease can be combined to generate a polynucleotide sequence which encodes for a polypeptide having desired characteristics. This method may be usefully applied to the generation of new antibodies or parts thereof having modified characteristics as compared to the parent antibody.
Inventors
- 👤 Carl Arne Krister Borrebaeck
- 👤 Ulf Hans Eskil Soderlind
- 👤 Rebecka Ingrid Camilla Ottosson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 10:36
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N15/1027; C12N15/1034; C12N15/1037; C12Q; C12Q1/00; C12Q1/68; C12Q1/6811; C12Q1/6844; C12Q1/6858
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
95 words
A method for generating a polynucleotide sequence or population of sequences from a parent polynucleotide sequence encoding one or more protein motifs, comprising the steps of a) digesting the parent polynucleotide sequence with an exonuclease to generate a population of fragments; b) contacting said fragments with a template polynucleotide sequence under annealing conditions; c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotide.
Claim 2
Independent Claim
95 words
A method for generating a polynucleotide sequence or population of sequences from a parent polynucleotide sequence encoding one or more protein motifs, comprising the steps of a) digesting the parent polynucleotide sequence with an exonuclease to generate a population of fragments; b) contacting said fragments with a template polynucleotide sequence under annealing conditions; c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotide.
Claim 3
Dependent Claim
32 words
A method according to claim 1 wherein the parent polynucleotide is double-stranded and the method further comprises the step of generating single-stranded polynucleotide sequence from said double-stranded fragments prior to step b).
Claim 4
Dependent Claim
16 words
A method according to claim 1 wherein the template polynucleotide sequence is the parent polynucleotide sequence.
Claim 5
Dependent Claim
16 words
A method according to claim 3 wherein the parent polynucleotide sequence has been subjected to mutagenesis.
Claim 6
Dependent Claim
19 words
A method according to claim 1 wherein the population of fragments generated in step b) is subjected to mutagenesis.
Claim 7
Dependent Claim
16 words
A method according to claim 4 or claim 5 wherein the mutagenesis is error prone mutagenesis.
Claim 8
Dependent Claim
11 words
A method according to claim 1 wherein the exonuclease is BAL31.
Claim 9
Dependent Claim
17 words
A method according to claim 1 wherein the parent polynucleotide sequence encodes an antibody or fragment thereof.
Claim 10
Dependent Claim
14 words
A method according to claim 1 wherein the parent polynucleotide sequence encodes an enzyme.
Claim 11
Dependent Claim
24 words
A method according to claim 1 further comprising the step of screening the at least one polynucleotide generated in step c) for desired characteristics.
Claim 12
Dependent Claim
29 words
A method according to claim 1 further comprising the step of expressing the at least one polynucleotide generated in step c) and screening the resulting polypeptide for desired characteristics.
Claim 13
Dependent Claim
33 words
A method for preparing a pharmaceutical composition which comprises, following the identification of a polynucleotide with desired characteristics by a method according to claim 1, adding said polynucleotide to a pharmaceutically acceptable carrier.
Claim 14
Dependent Claim
33 words
A method for preparing a pharmaceutical composition which comprises, following the identification of a polypeptide with desired characteristics by a method according to claim 11, adding said polypeptide to a pharmaceutically acceptable carrier.
Claim 15
Dependent Claim
34 words
A process which comprises, following the identification of a polynucleotide by a method according to claim 1, the manufacture of that polynucleotide, in whole or in part, optionally in conjunction with additional polynucleotide sequence.
Claim 16
Dependent Claim
33 words
A process which comprises, following the identification of a polypeptide by a method according to claim 11, the manufacture of that polypeptide, in whole or in part, optionally in conjunction with additional polypeptides.
Claim 17
Dependent Claim
15 words
A process according to claim 15 wherein the polypeptide is an antibody or fragment thereof.
Claim 18
Dependent Claim
12 words
A process according to claim 15 wherein the polypeptide is an enzyme.
Claim 19
Dependent Claim
40 words
A method of detecting and/or amplifying a target polynucleotide in a sample comprising contacting said sample with a polynucleotide having been identified by a method according to claim 1, said identified polynucleotide optionally being conjugated with an additional polynucleotide sequence.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-12-12 | 2004-06-12 | 2004-12-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-12-12 | 2008-06-12 | 2008-12-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-12-12 | 2012-06-12 | 2012-12-12 | $7700.00 | Window Closed / Lapsed |