A rapid method for detecting and mapping in vitro transcripts from supercoiied templates using endogenous RNase H

摘要

Transcription of DNA templates in nuclear extracts is a widely used technique to study the sequences and factors involved in transcription by RNA polymerase II. Two types of templates are commonly used: linear templates, where a single product accumulates extending from the initiation site to the end of the template, and supercoiied templates. If the RNA is radiolabeled specific transcripts formed from linear templates can be detected directly by gel electrophoresis followed by autoradiography. SinceRNA polymerase II does not terminate specifically there are not defined products formed by transcription of supercoiied templates by RNA polymerase II. Transcription from supercoiied templates can be assayed by either primer extension or nuclease protection assays, both of which require additional handling steps increasing the time required for die analysis and potentially introducing errors in recovery which can make quantitation more difficult. Since transcription from supercoiied templates is oftenmore efficient than transcription from linear templates (1, and references therein) and is more likely to reflect the physiological situation, transcription from sapercoiled templates is often the preferred assay.