Amplification of nucleic acid molecules

• US 5,169,766 A
• Filed: 06/14/1991
• Issued: 12/08/1992
• Est. Priority Date: 06/14/1991
• Status: Expired due to Fees

• Current Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Schuster, David M., Berninger, Mark S.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): KETTER, JAMES S
• Application Number:
  US07/715,603
• Time in Patent Office: 543 Days
• Field of Search: 435/91, 435/172.3, 435/172.1, 435/6, 435/193, 435/194, 536/27, 935/16, 935/77
• US Class Current: 435/91.2
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 2710/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

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.

Claims

1. 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.

2. The method of claim 1, wherein said promoter is a T7 promoter.

3. The method of claim 1, wherein said polymerase is a template-directed reverse transcriptase.

4. 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).

5. 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).

6. 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).

7. 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).

8. The method of claim 2, wherein said RNA polymerase is T7 RNA polymerase.

9. 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.

10. The kit of claim 9 which additionally contains a DNA polymerase.

11. The kit of claim 9 which additionally contains a RNA polymerase.

12. The kit of claim 11 wherein said RNA polymerase is T7 polymerase.

13. The kit of claim 10 wherein said DNA polymerase is a reverse transcriptase.