Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than thermus thermophilus DNA ligase.
To mimic large numbers of nicked DNA duplexes we used a technique that produces nicked duplex DNA substrates by hybridization of complementary oligonucleotides, adjacent to an initiating primer, which are ligated together by a thermostable DNA ligase. Sequential ligation of nonanucleotides to this primary duplex results in the formation of polymers that can be analyzed by gel electrophoresis. The extent of polymerization is a measure of the efficiency of ligation. We determined the efficiency of ligation of nonanucleotides, using various length initiating primers, with three thermostable DNA ligases: Thermus thermophilus (Tth), Thermus scotoductus (Ts), and Rhodothermus marinus (Rm). Analysis of the effect of temperature for each ligase, and for each directing primer length, revealed that at 37 and 41 degrees C there was variation between ligase efficiency in the order Rm > or = Ts > or = Tth. The higher temperature of 46 degrees C was optimal for polymerization with each of the ligases and Rm ligase was the most efficient. Analysis of directionality of the ligations reactions suggests that for each of the Thermus ligases we tested, there was a bias to polymerization of nonanucleotides in a 5'-3' direction.
Housby
J Nicholas
JN
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom. n.housby@oxagen.co.uk
Southern
Edwin M
EM
D011088:DNA Ligases / Q000032:analysis*; D017931:DNA Primers; D009693:Nucleic Acid Hybridization; D013045:Species Specificity; D013696:Temperature; D013824:Thermus / Q000201:enzymology*
D017931:DNA Primers; D011088:DNA Ligases