Amplification of nucleic acid molecules
AssigneeLife Technologies Corporation
InventorsDavid M. Schuster, Mark S. Berninger
Life of this patent
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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.
Claims13
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.
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.
Dependencies are inferred from the claim text.