Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions
AssigneeNew England Biolabs, Inc.
InventorsChristopher J. Noren, Paul D. Evans
Life of this patent
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Abstract
Multipurpose cloning in vitro phagemid vectors are disclosed, which vectors may be used to generate high specific-activity RNA probes. The multipurpose vectors also possess a bidirectional in vitro transcription system. Methods for constructing this system are also disclosed.
Claims15
A cloning/in vitro transcription vector comprising a vector engineered to have at least one polylinker, wherein the polylinker is flanked by opposing promoters, which promoters are recognized by a single species of RNA polymerase, and which have been modified by introduction of a first restriction endonuclease site into or proximate the first opposing promoter and a second restriction endonuclease site into or proximate the second re-opposing promoter to permit unidirectional transcription from one of the opposing promoters when the other opposing promoter has been rendered inoperable.
A method for determining the tolerance of a promoter for an introduced restriction endonuclease site comprising:
- (a) introducing the restriction site within or proximal to the promoter by site-directed mutagenesis;
- (b) assaying the promoter of step (a) for transcriptional activity;
- and
- (c) assaying the operability of the promoter of step (b) after cleavage by a restriction endonuclease which recognizes and cleaves the restriction site.
Dependencies are inferred from the claim text.