Nucleic Acids Research, 1978, Vol. 5, No. 5 1445-1464
© 1978
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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*
Department of Biophysical Sciences, University of Houston Houston, TX 77004, USA
Received February 27, 1978.
We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage 
b2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.
*Paper II in this series is reference 18.
+Present address: Department of Molecular Biophysics and Bio-chemistry, Yale University, New Haven, Connecticut 06510
++Present address: Department of Biology, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
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