RENATURATION OF BACTERIOPHAGE-LAMBDA DNA - DETERMINATION OF OPTIMAL RENATURATION CONDITIONS USING A SINGLE-STRAND-SPECIFIC DNASE AND ALKALINE-SUCROSE-GRADIENT ASSAY SYSTEM

Reannealed hybrid molecules of wild-type phage .lambda. DNA were prepared in aqueous solutions of formamide at a variety of NaCl concentrations at both room temperature (.apprxeq. 22.degree. C) and 37.degree. C. Treatment of the hybrid DNA molecules with the single-strand-specific nuclease S1 from Aspergillus oryzae followed by alkaline sucrose gradient sedimentation was used to monitor the extent and fidelity of hybridization. The optimal renaturation conditions at room temperature were 50% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 .mu.g DNA/ml. Optimal conditions at 37.degree. C were 32% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 .mu.g DNA/ml. Under these conditions approximately 85-90% of the input single-stranded DNA (molecular weight 1.5 .times. 107) was rendered S1-nuclease-resistant within 8 h at room temperature and 5 h at 37.degree. C. Neither Mg2+ nor spermidine appeared to have an effect on either the extent or fidelity of duplex formation. Experiments performed with excess enzyme and with .lambda./.lambda. imm 434 heteroduplex hybrids suggested that the hybrid DNA molecules formed under optimal conditions contained no, or only short (< 1%), mismatched regions.