Renaturation of Bacteriophage Lambda DNA. Determination of the Optimal Renaturation Conditions Using a Single-Strand-Specific DNase and Alkaline-Sucrose-Gradient Assay System
| Autor: | R. Wayne Davies, Thomas G. Fanning, P.H. Schreier |
|---|---|
| Rok vydání: | 1976 |
| Předmět: |
Formamide
Density gradient Sodium Chloride Nucleic Acid Denaturation Coliphages Biochemistry Nucleic acid thermodynamics chemistry.chemical_compound Centrifugation Density Gradient Centrifugation Nuclease Binding Sites Deoxyribonucleases Chromatography Aqueous solution biology Chemistry Osmolar Concentration Nucleic Acid Hybridization Molecular biology Kinetics DNA Viral Nucleic Acid Renaturation biology.protein DNA |
| Zdroj: | European Journal of Biochemistry. 62:173-179 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1976.tb10110.x |
| Popis: | Reannealed hybrid molecules of wild-type bacteriophage lambda DNA were prepared in aqueous solutions of formamide at a variety of NaCl concentrations at both room temperature ( 22 degrees C) and 37 degrees 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 found to be: 50% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 mug DNA/ml. Optimal conditions at 37 degrees C were: 32% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 mug DNA/ml. Under these conditions approximately 85-90% of the input single-stranded DNA (molecular weight 1.5 X 10(7)) was rendered S1-nuclease-resistant within 8 h at room temperature and 5 h at 37 degrees 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 that the hybrid DNA molecules formed under optimal conditions contained no, or only short (less than 1%), mismatched regions. |
| Databáze: | OpenAIRE |
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