Synthesis and Automated Detection of Fluorescently Labeled Primer Extension Products

Abstract

Common methods for determination of transcription initiation sites are based on the extension of radioactively labeled primers or internal labeling of extension products with reverse transcriptase. Here, we describe a modification of this procedure, adapted for automated detection of extension products by using fluorescent primers and an optimized reaction protocol. This facilitates high-throughput screening to detect mRNA start-points of large DNA regions and significantly reduces the time required to determine sites of transcription initiation.