Abstract: Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Main/Exploration

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{{Explor lien
   |wiki=    Sante
   |area=    MersV1
   |flux=    Main
   |étape=   Exploration
   |type=    RBID
   |clé=     ISTEX:95F72D731B104A5926D19504CA826E84B3E41529
   |texte=   Ligation of Eco RI endonuclease-generated DNA fragments into linear and circular structures
}}