US5169766A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Amplification of nucleic acid molecules
Assignee: Life Technologies Inc
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Filing: 1991-06-14
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Granted: 1992-12-08
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Expires: 2011-06-14
Abstract
Abstract A method for amplifying a nucleic acid molecule which employs a proto-promoter-containing nucleic acid molecule having a blocked 3' terminus. The invention also includes kits containing reagents for conducting the method.
Inventors
- 👤 David M. Schuster
- 👤 Mark S. Berninger
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 23:44
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6865; C12N2710/00; C12N2710/00011; C12N2710/20011; C12N2710/20022
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
301 words
A method of amplifying a desired nucleic acid molecule having a 3' hydroxyl terminus, which comprises the following steps in cycle: (A) incubating said desired nucleic acid molecule in the presence of a nucleic acid molecule comprising a proto-promoter, said proto-promoter being a single-stranded sequence region which, in double-stranded form is capable of mediating RNA transcription, said proto-promoter-containing nucleic acid molecule having a first region complementary to a sequence present in a 3' terminal region of said desired molecule, and a second region, located 5' to said first region, and containing said proto-promoter; said proto-promoter-containing nucleic acid molecule additionally having a 3' terminus that is incapable of being extended by a polymerase; said incubation being under conditions sufficient to permit nucleic acid hybridization to produce a hybridized double-stranded molecule comprising said first region of said proto-promoter-containing nucleic acid molecule and said 3' terminal region of said desired molecule; (B) incubating said hybridized molecule in the presence of an enzyme capable of extending the 3' terminus of said desired molecule in a template-dependent manner, to thereby convert said proto-promoter into a promoter capable of transcribing said desired molecule; (C) incubating said promoter in the presence of an RNA polymerase capable of promoter-dependent transcription of said desired molecule, to thereby produce a RNA molecule complementary to said desired nucleic acid molecule; (D) incubating said RNA molecule in the presence of a primer, said primer having a region complementary to a sequence present in a 3' terminal region of said RNA molecule; said incubation being under condition sufficient to permit nucleic acid hybridization to occur and to produce a hybridized double-stranded molecule comprising said primer and said 3' terminal region of said RNA molecule; and (E) extending said primer using a template-dependent polymerase, to thereby achieve the amplification of said desired nucleic acid molecule.
Claim 2
Independent Claim
301 words
A method of amplifying a desired nucleic acid molecule having a 3' hydroxyl terminus, which comprises the following steps in cycle: (A) incubating said desired nucleic acid molecule in the presence of a nucleic acid molecule comprising a proto-promoter, said proto-promoter being a single-stranded sequence region which, in double-stranded form is capable of mediating RNA transcription, said proto-promoter-containing nucleic acid molecule having a first region complementary to a sequence present in a 3' terminal region of said desired molecule, and a second region, located 5' to said first region, and containing said proto-promoter; said proto-promoter-containing nucleic acid molecule additionally having a 3' terminus that is incapable of being extended by a polymerase; said incubation being under conditions sufficient to permit nucleic acid hybridization to produce a hybridized double-stranded molecule comprising said first region of said proto-promoter-containing nucleic acid molecule and said 3' terminal region of said desired molecule; (B) incubating said hybridized molecule in the presence of an enzyme capable of extending the 3' terminus of said desired molecule in a template-dependent manner, to thereby convert said proto-promoter into a promoter capable of transcribing said desired molecule; (C) incubating said promoter in the presence of an RNA polymerase capable of promoter-dependent transcription of said desired molecule, to thereby produce a RNA molecule complementary to said desired nucleic acid molecule; (D) incubating said RNA molecule in the presence of a primer, said primer having a region complementary to a sequence present in a 3' terminal region of said RNA molecule; said incubation being under condition sufficient to permit nucleic acid hybridization to occur and to produce a hybridized double-stranded molecule comprising said primer and said 3' terminal region of said RNA molecule; and (E) extending said primer using a template-dependent polymerase, to thereby achieve the amplification of said desired nucleic acid molecule.
Claim 3
Dependent Claim
12 words
The method of claim 1, wherein said promoter is a T7 promoter.
Claim 4
Dependent Claim
13 words
The method of claim 1, wherein said polymerase is a template-directed reverse transcriptase.
Claim 5
Dependent Claim
29 words
The method of claim 1, wherein said desired nucleic acid molecule is single-stranded DNA, and wherein said method, in its entirety, is a cycle commencing with said step (A).
Claim 6
Dependent Claim
28 words
The method of claim 1, wherein said desired nucleic acid molecule is double-stranded DNA, and wherein said method, in its entirety, is a cycle commencing with step (A).
Claim 7
Dependent Claim
29 words
The method of claim 1, wherein said desired nucleic acid molecule is single-stranded RNA, and wherein said method, in its entirety, is a cycle commencing with said step (D).
Claim 8
Dependent Claim
29 words
The method of claim 1, wherein said desired nucleic acid molecule is double-stranded RNA, and wherein said method, in its entirety, is a cycle commencing with said step (D).
Claim 9
Dependent Claim
13 words
The method of claim 2, wherein said RNA polymerase is T7 RNA polymerase.
Claim 10
Dependent Claim
99 words
A kit for amplifying a desired nucleic acid molecule, said kit containing, in close compartmentalization, a first container which contains a nucleic acid molecule comprising a proto-promoter, said proto-promoter being a single-stranded sequence region which, in double-stranded form is capable of mediating RNA transcription and wherein said proto-promoter-containing nucleic acid molecule additionally possesses a 3' terminus that is incapable of being extended by a polymerase, and a second container which contains at least one primer molecule having a sequence region which is complementary to a sequence present in a 3' terminal region of a complement of the desired sequence.
Claim 11
Dependent Claim
99 words
A kit for amplifying a desired nucleic acid molecule, said kit containing, in close compartmentalization, a first container which contains a nucleic acid molecule comprising a proto-promoter, said proto-promoter being a single-stranded sequence region which, in double-stranded form is capable of mediating RNA transcription and wherein said proto-promoter-containing nucleic acid molecule additionally possesses a 3' terminus that is incapable of being extended by a polymerase, and a second container which contains at least one primer molecule having a sequence region which is complementary to a sequence present in a 3' terminal region of a complement of the desired sequence.
Claim 12
Dependent Claim
11 words
The kit of claim 9 which additionally contains a DNA polymerase.
Claim 13
Dependent Claim
11 words
The kit of claim 9 which additionally contains a RNA polymerase.
Claim 14
Dependent Claim
12 words
The kit of claim 11 wherein said RNA polymerase is T7 polymerase.
Claim 15
Dependent Claim
13 words
The kit of claim 10 wherein said DNA polymerase is a reverse transcriptase.
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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) | 1995-12-08 | 1996-06-08 | 1996-12-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-12-08 | 2000-06-08 | 2000-12-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-12-08 | 2004-06-08 | 2004-12-08 | $7700.00 | Window Closed / Lapsed |