Patent US4036816A successfully refreshed from Google Patents.
Patent US5756316A successfully refreshed from Google Patents.
US5756316A
Expired / Lapsed
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Molecular cloning by multimerization of plasmids
Assignee: Danisco US Inc
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Filing: 1995-11-02
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Granted: 1998-05-26
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Expires: 2015-11-02
Abstract
Abstract Novel methods of nucleic acid multimerization using PCR are provided, allowing direct transformation of cells and microorganisms.
Inventors
- 👤 Volker Schellenberger
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 11:10
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2414; C12N9/2417; C12N15/00; C12N15/09; C12N15/10; C12N15/63; C12N15/64; C12N15/66; C12N15/74; C12N15/75; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/686
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
96 words
A method of transforming a cell comprising: a) contacting i) a first linear nucleic acid comprising a first end sequence, a second end sequence and an intervening sequence; and ii) a second linear nucleic acid comprising a third end sequence homologous to said first end sequence and a fourth end sequence homologous to said second end sequence; to form a polymerase chain reaction mixture; b) subjecting said polymerase chain reaction mixture to conditions which permit amplification by the polymerase chain reaction for at least 3 cycles; c) transforming said cell with the product of step b).
Claim 2
Independent Claim
96 words
A method of transforming a cell comprising: a) contacting i) a first linear nucleic acid comprising a first end sequence, a second end sequence and an intervening sequence; and ii) a second linear nucleic acid comprising a third end sequence homologous to said first end sequence and a fourth end sequence homologous to said second end sequence; to form a polymerase chain reaction mixture; b) subjecting said polymerase chain reaction mixture to conditions which permit amplification by the polymerase chain reaction for at least 3 cycles; c) transforming said cell with the product of step b).
Claim 3
Dependent Claim
15 words
A method according to claim 1 wherein said first nucleic acid comprises a linearized plasmid.
Claim 4
Dependent Claim
22 words
A method according to claim 1 wherein said first end sequence and said second end sequence comprise a gene when ligated together.
Claim 5
Dependent Claim
13 words
A method according to claim 1 wherein said intervening sequence comprises a gene.
Claim 6
Dependent Claim
30 words
A method according to claim 1 wherein said first end sequence is identical to said fourth end sequence and said second end sequence is identical to said third end sequence.
Claim 7
Dependent Claim
27 words
A method according to claim 1 wherein said polymerase chain reaction mixture comprises a 100 fold excess of said second nucleic acid over said first nucleic acid.
Claim 8
Dependent Claim
23 words
A method according to claim 1 wherein said polymerase chain reaction mixture comprises roughly equal amounts of said first and second nucleic acids.
Claim 9
Dependent Claim
12 words
A method according to claim 1 wherein said cell is a microorganism.
Claim 10
Dependent Claim
13 words
A method according to claim 1 wherein said cell is a Bacillus species.
Claim 11
Dependent Claim
12 words
A method according to claim 1 wherein said cell is B. subtilis.
Claim 12
Dependent Claim
26 words
A method according to claim 1 further comprising the steps of d) selecting said transformed cell by selecting for the presence of the first nucleic acid.
Claim 13
Dependent Claim
28 words
A method according to claim 1 further comprising the steps of e) culturing said transformed cell under conditions which express a gene product of said first nucleic acid.
Claim 14
Dependent Claim
20 words
A method according to claim 12 further comprising the steps of f) isolating said gene product from said transformed cell.
Claim 15
Dependent Claim
35 words
A method according to claim 1 wherein prior to step a), a circular first nucleic acid is cleaved at the junction between said first and second end sequences to form a linear first nucleic acid.
Claim 16
Dependent Claim
48 words
A method according to claim 1 wherein said first nucleic acid is single-stranded, said first end sequence is capable of hybridizing to one strand of said homologous third end sequence, and said second end sequence is capable of hybridizing to one strand of said homologous fourth end sequence.
Claim 17
Dependent Claim
48 words
A method according to claim 1 wherein said second nucleic acid is single-stranded, said third end sequence is capable of hybridizing to one strand of said homologous first end sequence, and said fourth end sequence is capable of hybridizing to one strand of said homologous second end sequence.
Claim 18
Dependent Claim
44 words
A method according to claim 1, wherein said first and second nucleic acids are single stranded, and said first end sequence is capable of hybridizing to said third end sequence, and said second end sequence is capable of hybridizing to said fourth end sequence.
Claim 19
Independent Claim
87 words
A method of generating nucleic acid multimers comprising: a) contacting i) a first linear nucleic acid comprising a first end sequence, a second end sequence and an intervening sequence; and ii) a second linear nucleic acid comprising a third end sequence homologous to said first end sequence and a fourth end sequence homologous to said second end sequence; to form a polymerase chain reaction mixture; b) subjecting said polymerase chain reaction mixture to conditions which permit amplification by the polymerase chain reaction for at least 3 cycles.
Claim 20
Independent Claim
87 words
A method of generating nucleic acid multimers comprising: a) contacting i) a first linear nucleic acid comprising a first end sequence, a second end sequence and an intervening sequence; and ii) a second linear nucleic acid comprising a third end sequence homologous to said first end sequence and a fourth end sequence homologous to said second end sequence; to form a polymerase chain reaction mixture; b) subjecting said polymerase chain reaction mixture to conditions which permit amplification by the polymerase chain reaction for at least 3 cycles.
Claim 21
Dependent Claim
11 words
A nucleic acid multimer made by the method of claim 18.
Claim 22
Independent Claim
109 words
A method of making a chimeric nucleic acid comprising: a) contacting i) a first nucleic acid comprising a first end sequence, a second end sequence and an intervening sequence; and ii) a second nucleic acid comprising a third end sequence homologous to said first end sequence and a fourth end sequence homologous to said second end sequence; to form a polymerase chain reaction mixture, wherein the sequence of said third end sequence has at least one different nucleotide than the sequence of said first end sequence; and b) subjecting said polymerase chain reaction mixture to conditions which permit amplification by the polymerase chain reaction for at least 3 cycles.
Claim 23
Independent Claim
109 words
A method of making a chimeric nucleic acid comprising: a) contacting i) a first nucleic acid comprising a first end sequence, a second end sequence and an intervening sequence; and ii) a second nucleic acid comprising a third end sequence homologous to said first end sequence and a fourth end sequence homologous to said second end sequence; to form a polymerase chain reaction mixture, wherein the sequence of said third end sequence has at least one different nucleotide than the sequence of said first end sequence; and b) subjecting said polymerase chain reaction mixture to conditions which permit amplification by the polymerase chain reaction for at least 3 cycles.
Claim 24
Dependent Claim
21 words
A method according to claim 20 further comprising the step of c) transforming a cell with the product of step b).
Claim 25
Dependent Claim
32 words
A method according to claim 21 further comprising the step of d) culturing said transformed cell under conditions which permit the production of a chimeric gene product of said first nucleic acid.
Claim 26
Dependent Claim
20 words
A method according to claim 22 further comprising the steps of e) isolating said gene product from said transformed cell.
Claim 27
Dependent Claim
13 words
A method according to claim 21, wherein said cell is a Bacillus species.
Claim 28
Dependent Claim
13 words
A method according to claim 24, wherein said Bacillus species is B. subtilis.
Claim 29
Dependent Claim
12 words
The chimeric nucleic acid multimer made by the method of claim 20.
Claim 30
Independent Claim
130 words
A method of generating a library in a microorganism comprising: a) generating a plurality of first nucleic acids wherein said nucleic acids function as vectors each comprising a first end sequence, an intervening sequence, and a second end sequence, wherein said plurality of first nucleic acids comprise a population of library fragments, b) contacting said plurality of first nucleic acids with a second nucleic acid comprising third and fourth end sequences homologous to the first and second end sequences, respectively, of at least one of said first nucleic acids to form a polymerase chain reaction mixture; c) subjecting said polymerase chain reaction mixture to conditions which permit the polymerase chain reaction for at least 3 cycles; d) transforming said microorganism with the products of step c) thereby generating a library.
Claim 31
Independent Claim
130 words
A method of generating a library in a microorganism comprising: a) generating a plurality of first nucleic acids wherein said nucleic acids function as vectors each comprising a first end sequence, an intervening sequence, and a second end sequence, wherein said plurality of first nucleic acids comprise a population of library fragments, b) contacting said plurality of first nucleic acids with a second nucleic acid comprising third and fourth end sequences homologous to the first and second end sequences, respectively, of at least one of said first nucleic acids to form a polymerase chain reaction mixture; c) subjecting said polymerase chain reaction mixture to conditions which permit the polymerase chain reaction for at least 3 cycles; d) transforming said microorganism with the products of step c) thereby generating a library.
Claim 32
Dependent Claim
21 words
A method according to claim 27 wherein said library fragments are contained within the intervening sequence of said first nucleic acids.
Claim 33
Dependent Claim
17 words
A method according to claim 27 wherein said first and second end sequences comprise said library fragments.
Claim 34
Dependent Claim
18 words
A method according to claim 27 wherein said population of library fragments comprise a population of mutant genes.
Claim 35
Dependent Claim
21 words
A method according to claim 30 wherein said mutant genes are contained within the intervening sequence of said first nucleic acids.
Claim 36
Dependent Claim
17 words
A method according to claim 30 wherein said first and second end sequences comprise said mutant genes.
Claim 37
Dependent Claim
13 words
A method according to claim 30 wherein said gene is a protease gene.
Claim 38
Dependent Claim
11 words
A method according to claim 33 wherein said protease is subtilisin.
Claim 39
Dependent Claim
11 words
A method according to claim 30 wherein said gene is amylase.
Claim 40
Dependent Claim
10 words
A library made according to the method of claim 27.
Claim 41
Dependent Claim
8 words
A mutant library made according to claim 30.
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) | 2001-05-25 | 2001-11-24 | 2002-05-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-05-25 | 2005-11-24 | 2006-05-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-05-25 | 2009-11-24 | 2010-05-26 | $7700.00 | Window Closed / Lapsed |