Single-molecule DNA digestion by lambda-exonuclease

✍ Scribed by Johannes Dapprich

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 112 KB
• Volume: 36
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19990701)36:3<163::aid-cyto3>3.0.co;2-r

✦ Synopsis

We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained -DNA attached to optically trapped beads. The setup has been described previously (Dapprich and Nicklaus: Bioimaging 6:25-32, 1998) and works by observing the change in position of a trapped bead depending on its viscous drag force during motion. The drag force of a naked bead increases with each attached DNA molecule to a characteristic level that depends on the length and the number of DNAs per bead. A single undigested DNA molecule on a bead will remain stable for extended periods and exhibit a constant drag force in flow. If -exonuclease is added, the drag force decreases from the level for one strand of DNA on a bead to that of a naked bead in about 45 min. This result indicates that the digestion of native -DNA by -exonuclease occurs at an average rate of approximately 15-20 Hz.