Abstract
An important manipulation in molecular genetics is to make ordered deletions into a cloned piece of DNA. The most widely used application of this method is in DNA sequencing. Ordered deletions can also be used in delineating sequences that are important for the function of a gene, such as those required for transcription. The principle behind using deletions for sequencing is that consecutive parts of a fragment cloned into a plasmid vector are brought adjacent to a sequencing primer site in the vector. Deletions are generated by digesting DNA unidirectionally with Escherichia coli exonuclease III (ExoIII) (1). ExoIII digests one strand of double-stranded DNA by removing nucleotides from 3′ ends if the end is blunt or has a 5′ protrusion. A 3′ protrusion of 4 bases or more is resistant to ExoIII digestion.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Henikoff, S. (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351–359.
Henikoff, S. (1990) Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates. Nucleic Acids. Res. 18, 2961–2966.
Nakayama, K. and Nakauchi, H. (1989) An Improved method to make sequential deletion mutants for DNA sequencing. Trends Genet. 5, 325.
Steggles, A. W. (1989) A rapid procedure for creating nested sets of deletions using mini-prep plasmid DNA samples. Biotechniques 7, 241,242.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Putney, S. D., Benkovic, S. J., and Schimnel, P. R. (1981) A DNA fragment with an α-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc. Natl. Acad. Sci. USA 78, 7350–7354.
Henikoff, S. (1987) Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 155, 156–165.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc.
About this protocol
Cite this protocol
Clark, D., Henikoff, S. (1996). Ordered Deletions Using Exonuclease III. In: Trower, M.K. (eds) In Vitro Mutagenesis Protocols. Methods In Molecular Medicine™, vol 57. Humana Press. https://doi.org/10.1385/0-89603-332-5:139
Download citation
DOI: https://doi.org/10.1385/0-89603-332-5:139
Publisher Name: Humana Press
Print ISBN: 978-0-89603-332-0
Online ISBN: 978-1-59259-544-0
eBook Packages: Springer Protocols