Automated fluorescence-based restriction fragment analysis.

Abstract

We describe a procedure for the automated analysis of DNA restriction fragments in an agarose gel matrix. The procedure is based upon simultaneously digesting DNA with restriction enzymes and ligating a fluorochrome-labeled linker to the cut ends. The fluorochrome-labeled restriction fragments are loaded on a horizontal agarose gel scanning system (Applied Biosystems Model 362 GENE SCANNER Fluorescent Fragment Analyzer) for electrophoresis and data analysis. Mobility parameters for DNA fragment migration and detection sensitivity for the instrument are described. In addition, we demonstrate some unique applications, including electrophoresis and fragment size conversion of up to 72 cosmid restriction digests in 4 h, applicability of the system for subsequent DNA transfers and hybridizations, utility for establishing overlapping restriction maps of cosmids, and discrimination of restriction digest specific fragment ends following a double-digest protocol.