AUTOMATED FLUORESCENCE-BASED RESTRICTION FRAGMENT ANALYSIS

We describe a procedure for the automated analysis of DNA restriction fragments in an agarose gel matrix. The procedure is based upon simult aneously digesting DNA with restriction enzymes and ligating a fluoroc hrome-labeled linker to the cut ends. The fluorochrome-labeled restric tion fragments are loaded on a horizontal agarose gel scanning system (Applied Biosystems Model 362 GENE SCANNER(TM) Fluorescent Fragment An alyzer) for electrophoresis and data analysis. Mobility parameters for DNA fragment migration and detection sensitivity for the instrument a re described In addition, we demonstrate some unique applications, inc luding electrophoresis and fragment size conversion of up to 72 cosmid restriction digests in 4 h, applicability of the system for subsequen t DNA transfers and hybridizations, utility for establishing overlappi ng restriction maps of cosmids, and discrimination of restriction dige st specific fragment ends following a double-digest protocol.