US4666839A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods and materials for obtaining microbial expression of polypeptides including bovine prolactin
Assignee: Amgen Inc
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Filing: 1982-12-01
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Granted: 1987-05-19
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Expires: 2002-12-01
Abstract
Abstract Disclosed are methods for selective modification of double-stranded DNA sequences facilitating their storage and incorporation into expression vectors. Manufactured partially double-stranded DNA primers are hybridized to complementary regions of single-stranded forms of DNA sequences to be altered. Desired, selectively modified double-stranded DNA sequences are then formed by DNA polymerization and specific nuclease digestion of undesired double- and single-stranded regions. Illustratively provided are DNA sequences useful in the microbial expression of bovine prolactin and other valuable polypeptides such as avian growth hormone.
Inventors
- 👤 Lawrence M. Souza
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 23:23
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/57554; C07K14/61
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
232 words
A method for selective modification of a double-stranded DNA sequence, comprising the steps: (1) forming a first selected single-stranded DNA copy of said double-stranded DNA sequence; (2) hybridizing said first selected single-stranded copy to a manufactured DNA sequence which comprises (a) a single-stranded portion complementary to a selected portion of said first selected single-stranded copy, and (b) a double-stranded portion having a free end and an end wherein one strand is joined to the single-stranded portion, which double-stranded portion includes base pairs forming a recognition site for cleavage by a restriction endonuclease enzyme or includes at its free end base pairs forming half of a recognition site for cleavage by a blunt-end-forming restriction endonuclease; (3) incubating the hybridized product of step (2) with nucleotide triphosphates and DNA polymerase under conditions facilitative of 3' extension of the single-stranded portion of said manufactured sequence with bases complementary to said first selected single-stranded DNA copy, so that said double-stranded DNA sequence is reconstructed except for that portion of its sequence 3' to the site of hybridization of the selected single-stranded copy to the 5' end of the single-stranded portion of said manufactured sequence; (4) treating the product of step (3) to delete single-stranded sequences 3' to the site of hybridization of the selected single-stranded copy to the 5' end of the single-stranded portion of said manufactured sequence; and (5) recovering the modified double-stranded DNA sequence.
Claim 2
Independent Claim
232 words
A method for selective modification of a double-stranded DNA sequence, comprising the steps: (1) forming a first selected single-stranded DNA copy of said double-stranded DNA sequence; (2) hybridizing said first selected single-stranded copy to a manufactured DNA sequence which comprises (a) a single-stranded portion complementary to a selected portion of said first selected single-stranded copy, and (b) a double-stranded portion having a free end and an end wherein one strand is joined to the single-stranded portion, which double-stranded portion includes base pairs forming a recognition site for cleavage by a restriction endonuclease enzyme or includes at its free end base pairs forming half of a recognition site for cleavage by a blunt-end-forming restriction endonuclease; (3) incubating the hybridized product of step (2) with nucleotide triphosphates and DNA polymerase under conditions facilitative of 3' extension of the single-stranded portion of said manufactured sequence with bases complementary to said first selected single-stranded DNA copy, so that said double-stranded DNA sequence is reconstructed except for that portion of its sequence 3' to the site of hybridization of the selected single-stranded copy to the 5' end of the single-stranded portion of said manufactured sequence; (4) treating the product of step (3) to delete single-stranded sequences 3' to the site of hybridization of the selected single-stranded copy to the 5' end of the single-stranded portion of said manufactured sequence; and (5) recovering the modified double-stranded DNA sequence.
Claim 3
Dependent Claim
64 words
The method according to claim 1 further comprising the steps: (6) forming a second selected single-stranded DNA copy of said modified double-stranded DNA sequence of step (5), said second selected single-stranded DNA copy being the partial complement of said first selected single-stranded copy of step (1); and (7) repeating steps (2), (3), (4), and (5) on said second selected single-stranded copy of step (6).
Claim 4
Dependent Claim
21 words
The method according to claim 1 wherein said double-stranded portion of said manufactured DNA sequence includes a sequence coding for ##STR33##
Claim 5
Dependent Claim
24 words
The method according to claim 1 wherein said first selected single-stranded copy of step (1) is the plus strand of said double-stranded DNA sequence.
Claim 6
Dependent Claim
24 words
The method according to claim 1 wherein said first selected single-stranded copy of step (1) is the minus strand of said double-stranded DNA sequence.
Claim 7
Dependent Claim
45 words
The method according to claim 1 wherein said 5' end of said double-stranded portion of said manufactured DNA sequence comprises bases providing the 3' end of a blunt-ended restriction site selected from the group consisting of: PvuII, HpaI, BalI, SmaI, HaeIII, AluI, NaeI, and StuI.
Claim 8
Dependent Claim
39 words
The method according to claim 1 wherein said manufactured DNA sequence comprises a range of from about 10 to about 20 bases forming said single-stranded portion and from about 10 to about 20 base pairs forming said double-stranded portion.
Claim 9
Dependent Claim
22 words
The method according to claim 1 wherein said first selected single-stranded DNA copy is formed by denaturation of said double-stranded DNA sequence.
Claim 10
Dependent Claim
20 words
The method according to claim 1 wherein said first selected single-stranded DNA copy is formed by a single-stranded filamentous bacteriophage.
Claim 11
Dependent Claim
20 words
The method according to claim 2 wherein said first selected single-stranded DNA copy is formed by a single-stranded filamentous bacteriophage.
Claim 12
Dependent Claim
20 words
The method according to claim 10 wherein said second selected single-stranded DNA copy is formed by a single-stranded filamentous bacteriophage.
Claim 13
Dependent Claim
19 words
The method according to claim 1 wherein S1 nuclease is employed in said treatment for deletion of single-stranded regions.
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) | 1990-05-18 | 1990-11-17 | 1991-05-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-05-18 | 1994-11-17 | 1995-05-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-05-18 | 1998-11-17 | 1999-05-19 | $7700.00 | Window Closed / Lapsed |