Biochemical Method for Mapping Mutatio: Nuclease: The Location of Deletions and 1

S1 nuclease (EC 3.1.4.X), a single-strand- specific nuclease, can be used to accurately map the loca- tion of mutational alterations in simian virus 40 (SV40) DNA. Deletions of between 32 and 190 base pairs, which are at or below the limit of detectability by conventional electron microscopic analysis of heteroduplex DNAs, have been located in this way. To map a deletion, a lixture of unit length, linear DNA, prepared from the SV40 deletion mutant and its wild-type parent, are denatured and re- annealed to form heteroduplexes. SI nuclease can cut such heteroduplexes at the nonbase-paired region to produce fragments whose lengths correspond to the position of the deletion. Similarly, specific fragments are produced when S1 nuclease cleaves a heteroduplex formed from the DNAs of SV40 temperature-sensitive mutants and either their revertants or wild-type parents. Thus, the positions of the nonhomology between these DNAs can be determined. S1 nuclease (EC 3.1.4.X) from Aspergillus oryzae degrades single- but not double-stranded DNA (1); nevertheless, super- helical simian virus 40 (SV 40) (2) and polyoma (3) INAs are converted to unit length linear molecules by this enzyme. Presumably, this occurs because unpaired, or weakly hydro- gen-bonded regions, susceptible to S1 nuclease, occur or can be induced in the strained superhelical molecule. Since nicked circular molecules appeared to be intermediates in the double- strand cleavage, we surmised that the enzyme was capable of cleaving the intact strand opposite or near the nick (2). This property suggested that S1 nuclease could be used to map the location of small deletions, insertions, or, in fact, any difference in base sequence between otherwise homologous