US5605793A
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US
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Methods for in vitro recombination
Assignee: Codexis Inc
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Filing: 1994-02-17
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Granted: 1997-02-25
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Expires: 2014-02-17
Abstract
Abstract A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro 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 of proteins.
Inventors
- 👤 Willem P. C. Stemmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 20:43
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: 6
Claim 1
Independent Claim
181 words
A method for forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, comprising: a) adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotide, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other thereby forming a mutagenized double-stranded polynucleotide; and d) repeating steps b) and c) for at least two further cycles, wherein the resultant mixture in step (b) of a further cycle includes the mutagenized double-stranded polynucleotide from step c) of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide.
Claim 2
Independent Claim
181 words
A method for forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, comprising: a) adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotide, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other thereby forming a mutagenized double-stranded polynucleotide; and d) repeating steps b) and c) for at least two further cycles, wherein the resultant mixture in step (b) of a further cycle includes the mutagenized double-stranded polynucleotide from step c) of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide.
Claim 3
Dependent Claim
33 words
The method of claim 1 wherein the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA.
Claim 4
Dependent Claim
18 words
The method of claim 1 wherein the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides.
Claim 5
Dependent Claim
21 words
The method of claim 1 wherein the size of the double-stranded random fragments is from about 5 bp to 5 kb.
Claim 6
Dependent Claim
17 words
The method of claim 1, wherein step d) comprises repeating steps b)-c) for at least 10 cycles.
References Cited (Backward Citations)
US4994368A
Cited
US5043272A
Cited
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