This chapter deals with specific labeling of 3' termini with T4 DNA polymerase. T4 DNA polymerase is both a 5' → 3' polymerase and a 3' → 5' exonuclease.The enzyme, T4 DNA polymerase, has been proven useful in analyzing and manipulating terminal DNA sequences because its rate of polymerization is greater than its rate of exonucleolytic hydrolysis. The combined polymerase and exonuclease activities of the T4 DNA polymerase have suggested several applications. Radioactivity can be introduced at the 3' terminus of a duplex without altering the structure of the terminus. One or more nucleotides can be removed from a 3' terminus at either a nick or at the end of a duplex molecule. The extent of removal can be precisely predicted if previous sequence data exists. Similarly, one or more nucleotides can be added to a 3' terminus which is base-paired to an appropriate template. The chapter also presents the reaction conditions.
ASJC Scopus subject areas
- Molecular Biology