UNIT 7.4B Dideoxy DNA Sequencing with Chemiluminescent Detection
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Martin, C. S. 2001. Dideoxy DNA Sequencing with Chemiluminescent Detection. Current Protocols in Molecular Biology. 47:7.4B:7.4B.1–7.4B.14.
- Published Online: 1 MAY 2001
- Published Print: JUL 1999
Standard dideoxy DNA sequencing can be performed easily and efficiently with nonisotopic, chemiluminescent detection by utilizing primers labeled with biotin in the sequencing reactions. As described in this unit, reaction products are separated by denaturing gel electrophoresis, transferred to a nylon membrane, and detected by first binding a streptavidin-alkaline phosphatase conjugate, then incubating with a chemiluminescent 1,2-dioxetane substrate. The emitted light signal is imaged on standard X-ray film, producing high-resolution DNA sequencing ladders. Indirect alkaline phosphatase-labeling of biotinylated DNA with free streptavidin and biotinylated alkaline phosphatase is also detailed, Finally, the detection of sequencing reactions labeled with other haptens using specific antibody-alkaline phosphatase conjugates is described.