Digestion conditions resulting in altered cut site specificity for HinfI.

Analysis of DNA samples for restriction fragment length polymorphisms requires correct recognition of the normal restriction sites. Some restriction endonucleases have been shown to exhibit altered cut site specificity (star activity) under certain reaction conditions (1, 2). We report here the results of experiments demonstrating Hinfi star activity. Two ngs of pUC18 DNA or SV40 DNA were digested overnight with Hinfi purchased from New England Biolabs (NEB), Beverly, MA; Bethesda Research Labs (BRL), Gaithersburg, MD; or Promega, Madison, WI in 40 y\ of 100 mM NaCl, 10 raM Tris-HCl (pH 7.4), 10 mM MgCl2, 5 mM /3-Mercaptoethanol (BME), and 100 ̂ g/ml bovine serum albumin (BSA). Four /igs of human genomic DNA, extracted from blood according to standard procedures, were digested with Hinfi in a final volume of 80 /xl under the conditions stated above. Final concentrations of Hinfi storage buffer [50 mM KC1, 10 mM TrisHCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 200 /tg/ml BSA, and 50% glycerol], glycerol, BSA, NaCl, KC1, and DTT as well as digestion time were varied. Hinfi digested DNA samples were electrophoresed in agarose gels. Human DNA samples were transferred to a nylon membrane according to the procedure of Southern and hybridized with the four single-locus probes MSI, MS31, MS43, and g3 (3). Relaxed cut site specificity, as indicated by the appearance of additional bands on ethidium bromide stained gels (pUC18 and SV40 DNA) and autoradiographs (genomic DNA), is observed with Hinfi from all manufacturers tested under certain digestion conditions. Additional bands are faintly visible in Hinfi digested pUC18 DNA using 80 or 160 units of enzyme with 2.5% glycerol or 320 units of enzyme and 1.25% glycerol. The effect of high enzyme and glycerol concentrations on relaxation of Hinfi specificity is enhanced by increasing digestion times. When pUC18 is digested with 80 units of Hinfi at 2.5% glycerol, additional bands appear at 16 hours of incubation and become more intense as digestion times are increased up to two days. At 200 units of enzyme and 25% glycerol, extra bands are visible at 4 hours of digestion and increase in intensity with longer incubation times. Varying the concentrations of KC1 (10 mM to 200 mM), NaCl (10 mM to 200 mM), BSA (0.4 /tg or 4 /tg), and DTT (0.05 mM or 0.5 mM) in the reaction buffer did not affect Hinfi specificity.