A Universal Nested Deletion Method Using an Arbitrary Primer and Elimination of a Unique Restriction Site
The generation of nested deletions within cloned fragments of DNA has important applications in molecular biology. For DNA sequencing, nested deletions provide a series of overlapping templates that can be used to generate a composite sequence with a single sequencing primer; in gene and protein functional studies, nested deletions allow researchers to locate and define a variety of functional domains based on regions of deletions.
KeywordsVortex Glycerol Phenol EDTA Electrophoresis
- 1.Legerski, R. J., Hodnett, J. L., and Gray, H. B., Jr. (1978) Extracellular nucleases of Pseudomonas BAL 31 III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acids Res. 5, 1445.PubMedCrossRefGoogle Scholar
- 10.Protocol for Transformer™ Site-Directed Mutagenesis Kit. (1994) Clontech Laboratories, Inc, PT1130-1, Palo Alto, CA.Google Scholar
- 11.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
- 12.Zhu, L. (1992) Highly efficient site-directed mutagenesis of dsDNA plasmids. CLONTECHniques VII(1), 1–5.Google Scholar
- 13.Masumune, Y. and Richardson, C. A. (1971) Strand displacement during deoxyribonucleic acid synthesis at single-strand breaks. J. Biol. Chem. 246. 2692–2701.Google Scholar