Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.
We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible.
D001230:Aspergillus / Q000201:enzymology; D004269:DNA, Bacterial / Q000032:analysis* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008851:Micropore Filters; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D011513:Proteus mirabilis / Q000032:analysis; D012965:Sodium Chloride / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012965:Sodium Chloride; D004720:Endonucleases; D015032:Zinc