Abstract
There are two basic enzymatic activities that are used to end-label DNA with radioactive phosphate (32P). The enzyme T4-polynucleotide kinase will use the substrate ATP to add a phosphate group (the gamma-phosphate of the ATP molecule) preferentially to the 5′ ends of the molecule. The enzyme DNA polymerase will “fill-in” recessed 3′ ends with the complimentary nucleotides, and can also create recessed 3′ ends from “blunt” ends or even 3′ overhanging ends by its 3′–5′ exonuclease activity, then fill in such ends as well. However, the polymerase enzyme usually used for the fill-in reaction, the Klenow fragment of E. coli DNA Pol I, has very low 3′–5′ exonuclease activity, and therefore its fill-in activity on blunt and 3′ overhanging ends could in principle be ignored for the purpose of end-labeling DNA for sequencing (see Note 1).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Maxam, A. M and Gilbert, W. (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Method. Enzymol 65, 499–560
Maniatis, T, Fritsch, E F., and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1993 Humana Press Inc. Totowa, New Jersey
About this protocol
Cite this protocol
Pichersky, E. (1993). Terminal Labeling of DNA for Maxam and Gilbert Sequencing. In: Griffin, H.G., Griffin, A.M. (eds) DNA Sequencing Protocols. Methods in Molecular Biology™, vol 23. Humana Press. https://doi.org/10.1385/0-89603-248-5:247
Download citation
DOI: https://doi.org/10.1385/0-89603-248-5:247
Publisher Name: Humana Press
Print ISBN: 978-0-89603-248-4
Online ISBN: 978-1-59259-510-5
eBook Packages: Springer Protocols