Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the beh avior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-pr otein interactions, such as the DNA digestion reaction by lambda exonucleas e. In conventional methods it is impossible to analyze the dynamics of an i ndividual lambda exonuclease molecule on a DNA because they can only observ e the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lam bda exonuclease, a stained lambda DNA molecule with one biotinylated termin al was fixed on an avidin-coated coverslip and straightened using a d.c. el ectric field. Microscopic observation of digestion of a straightened DNA mo lecule by lambda exonuclease revealed that the DNA digestion rate was simil ar to 1000 bases/s and also demonstrated high processivity.