Open AccessBiochemical procedure for production of small deletions in simian virus 40 DNA.
Author(s) -
John Carbon,
Thomas Shenk,
Paul Berg
Publication year - 1975
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.72.4.1392
Subject(s) - biology , hpaii , cleavage (geology) , exonuclease , restriction enzyme , microbiology and biotechnology , ecori , dna , in vitro recombination , endonuclease , mutant , exonuclease iii , genetics , gene , dna polymerase , complementary dna , molecular cloning , escherichia coli , paleontology , gene expression , fracture (geology) , dna methylation
A simple biochemical procedure for producing small deletions (15 to 50 base pairs) at virtually any location in simian virus 40 DNA has been developed. The steps involved are: cleavage of the closed-circular DNA to produce a linear structure followed by 5'-exonuclease digestion to expose a short single-stranded segment at each 3' end of the molecule. Mutants containing deletions at the site of the cleavage are obtained by infecting permissive monkey kidney cells with the exonuclease-treated DNA in the presence or absence of a helper DNA (depending upon whether or not the site of cleavage and therefore the deletion occurred in a gene required for vegetative multiplication). In this paper viable mutants with deletions at the HpaII endonuclease cleavage site (0.735 map position) and defective trans-complementable mutants with deletions at the EcoRI endonuclease cleavage site (0/1.0 map position) were isolated.