Patent US6153410A successfully refreshed from Google Patents.
US6153410A
Expired / Lapsed
US
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Recombination of polynucleotide sequences using random or defined primers
Assignee: California Institute of Technology
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Filing: 1997-08-04
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Granted: 2000-11-28
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Expires: 2017-08-04
Abstract
Abstract A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.
Inventors
- 👤 Frances H. Arnold
- 👤 Zhixin Shao
- 👤 Joseph A. Affholter
- 👤 Huimin H Zhao
- 👤 Lorraine J. Giver
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 03:25
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/102; C12N15/1027; C12N9/00; C12N9/14; C12N9/16; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C12N9/78; C12N9/80; C12Q; C12Q1/00; C12Q1/68; C12Q1/6811; C12Q1/6813; C12Q1/6827; C12Q1/683
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
232 words
A method of evolving a polynucleotide toward acquisition of a desired property, comprising: a) contacting at least one template polynucleotide with a set of defined-sequence primers, the set of defined-sequence primers comprising a plurality of both forward and reverse primers; b) conducting a multi-cycle polynucleotide extension reaction on the at least one template polynucleotide and the set of defined-sequence primers, wherein: (i) in at least one cycle, the primers anneal to the at least one template polynucleotide and prime replication of the at least one template polynucleotide thereby generating a pool comprising overlapping fragments which are shorter in length than the at least one template polynucleotide and which overlap to span the at least one template polynucleotide; and (ii) in at least one subsequent cycle, the overlapping fragments generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations formiing annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form a further pool of overlapping fragments; whereby the multi-cyclic polynucleotide extension reaction is continued for sufficient cycles until the further pool of overlapping fragments includes variant forms of the at least one template polynucleotide; and c) screening or selecting the variant forms of the at least one template polynucleotide, or expression products thereof, for an altered or enhanced property relative to the at least one template polynucleotide or an expression product thereof.
Claim 2
Independent Claim
232 words
A method of evolving a polynucleotide toward acquisition of a desired property, comprising: a) contacting at least one template polynucleotide with a set of defined-sequence primers, the set of defined-sequence primers comprising a plurality of both forward and reverse primers; b) conducting a multi-cycle polynucleotide extension reaction on the at least one template polynucleotide and the set of defined-sequence primers, wherein: (i) in at least one cycle, the primers anneal to the at least one template polynucleotide and prime replication of the at least one template polynucleotide thereby generating a pool comprising overlapping fragments which are shorter in length than the at least one template polynucleotide and which overlap to span the at least one template polynucleotide; and (ii) in at least one subsequent cycle, the overlapping fragments generated in a previous cycle are denatured to single-stranded fragments, which anneal in new combinations formiing annealed fragments, whereby one strand of an annealed fragment primes replication of the other to form a further pool of overlapping fragments; whereby the multi-cyclic polynucleotide extension reaction is continued for sufficient cycles until the further pool of overlapping fragments includes variant forms of the at least one template polynucleotide; and c) screening or selecting the variant forms of the at least one template polynucleotide, or expression products thereof, for an altered or enhanced property relative to the at least one template polynucleotide or an expression product thereof.
Claim 3
Dependent Claim
31 words
The method of claim 1, wherein the at least one template polynucleotide is a plurality of different template polynucleotides, and the variant forms comprise recombinant forms of the different template polynucleotides.
Claim 4
Dependent Claim
15 words
The method of claim 2, wherein the plurality of different template polynucleotides are allelic variants.
Claim 5
Dependent Claim
45 words
The method of claim 1, wherein at least some of the pool of overlapping fragments generated in the at least one cycle of step (i) differ from each other due to priming from a primer lacking perfect complementarity with the at least one template polynucleotide.
Claim 6
Dependent Claim
18 words
The method of claim 1, further comprising amplifiing the variant forms of the at least one template polynucleotide.
Claim 7
Dependent Claim
27 words
The method of claim 1, wherein the polynucleotide extension is conducted under conditions of incomplete extension of the primers hybridized to the at least one template polynucleotide.
Claim 8
Dependent Claim
25 words
The method of claim 1, wherein the primers hybridized to the at least one template polynucleotide are extended by fewer than 300 nucleotides on average.
Claim 9
Dependent Claim
19 words
The method of claim 1, wherein the primers in the set of defined-sequence primers are 6-100 nucleotides in length.
Claim 10
Dependent Claim
25 words
The method of claim 1, wherein at least one defined-sequence primer further comprises a random nucleotide at one or more nucleotide positions with the primer.
Claim 11
Dependent Claim
23 words
The method of claim 1, wherein the at least one template polynucleotide is removed after the at least one cycle of step (i).
Claim 12
Dependent Claim
15 words
The method of claim 1, wherein the at least one template polynucleotide is single stranded.
Claim 13
Dependent Claim
51 words
The method of claim 1, wherein the at least one cycle of step (i) is performed under conditions of incomplete extension of the primers hybridized to the at least one template polynucleotide, and the method further comprises adjusting the conditions to allow complete extension of the annealed fragments in step (ii).
Claim 14
Dependent Claim
29 words
The method of claim 1, wherein the primers in the primer set are exhausted during the polynucleotide extension reaction thereby forcing annealing of the overlapping fragments in step (ii).
Claim 15
Dependent Claim
22 words
The method of claim 1, wherein the average length of the overlapping fragments in the polynucleotide extension reaction increases with successive cycles.
Claim 16
Dependent Claim
16 words
The method of claim 1, wherein the at least one template polynucleotide is of unknown sequence.
Claim 17
Dependent Claim
21 words
The method of claim 1, wherein the variant forms of the at least one template are screened for an enzymatic activity.
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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) | 2003-11-28 | 2004-05-29 | 2004-11-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-11-28 | 2008-05-29 | 2008-11-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-11-28 | 2012-05-29 | 2012-11-28 | $7700.00 | Window Closed / Lapsed |