UNIT 7.4A DNA Sequencing by the Dideoxy Method
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Slatko, B. E., Albright, L. M., Tabor, S. and Ju, J. 2001. DNA Sequencing by the Dideoxy Method. Current Protocols in Molecular Biology. 47:7.4A:7.4A.1–7.4A.39.
- Published Online: 1 MAY 2001
- Published Print: JUL 1999
In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single-stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S-labeled. The reaction also contains one of four dideoxyribonucleoside triphosphates (ddNTPs), which terminate elongation when incorporated into the growing DNA chain. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high-resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. Three variations of the dideoxy sequencing procedure are currently in use and are presented in this unit. In the “labeling/termination“ procedure, primer chains are initially extended and labeled in the absence of terminating ddNTPs, whereas in the traditional “Sanger” procedure, labeling and termination of primer chains occur in a single step. A recent variation of the dideoxy sequencing method is thermal cycle sequencing in which the reaction mixture, containing template DNA, primer, thermostable DNA polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation, annealing, and elongation steps. The resulting linear amplification of the sequencing products allows much less template DNA to be used and eliminates independent primer annealing and template denaturation steps, which are required for the labeling/termination or Sanger procedures. The use of automated fluorescent sequencers ofr four-color dideoxy DNA sequencing is also described in detail.