SINGLE-MOLECULE ANALYSIS OF RESTRICTION DNA FRAGMENTS USING FLUORESCENCE CORRELATION SPECTROSCOPY

The cleavage of fluorescence-labeled M13DNA (7250 bp) using HaeIII, Hg aI, BsmAI, and BspMI was analyzed by fluorescence correlation spectros copy (FCS) in a small volume (1.5 x 10(-15) liters). The digestion pro cess can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived a practical equation for estimating the number of molecules in the FCS measurements. Under standard enzymatic conditions, HaeIII and BsmAI digested fluorescence-labeled DNA to completion in the range of 8 h, whereas HgaI and BspMI digested the DNA after 40 h. The comparis on of recognition sequences suggested that some tagged nucleotides cou ld be inserted between the recognition site and the cleavage site of t he slow enzyme group. The decrease in amplitude in the fluorescence co rrelation function quantitatively monitors the hydrolysis of DNA durin g the digestion process. (C) 1998 Academic Press.