Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus and Rhodothermus marinus DNA ligases to other thermophilic ligases

Abstract

We describe the characterisation of four thermo-stable NAD(+)-dependent DNA ligases, from Thermus thermophilus (Tth), Thermus scotoductus (Ts), Rhodothermus marinus (Rm) and Thermus aquaticus (Taq), by an assay which measures ligation rate and mismatch discrimination. Complete libraries of octa-, nona- and decanucleotides were used as substrates. The assay comprised the polymerisation of oligo-nucleotides initiated from a 17 base 'primer', using M13mp18 ssDNA as template. Polymers of ligation products were analysed by polyacrylamide gel electro-phoresis. Under optimum conditions, the enzymes produced polymers ranging from 8 to 16 additions; there was variation between enzymes and the length of the oligonucleotides had a strong effect. The optimal total oligonucleotide concentration for each library was approximately 4 nmol. We compared the rates of ligation between the four ligases using an octanucleotide library as substrate. By this criterion, the Ts and Rm ligases are far more active compared to the more commonly available thermostable ligases.

Figure 1

Ligation of a library of oligonucleotides to M13mp18 ssDNA. The primer, M13P, was used to initiate ligation of oligonucleotides from a complete library. The ‘star’ represents the ³²P radiolabel. The library was chemically phosphorylated (see Materials and Methods) and each complementary oligonucleotide sequentially ligated as shown. The ligation reaction is depicted to proceed in either of two directions although we have previously demonstrated, for Tth, that the reaction goes predominantly in the 3′→5′ direction (13), i.e. away from the multiple cloning site.

Figure 2

Effect of the concentration of octa-, nona- and decanucleotide library on the rate of ligation by Tth. (A) The image is the result of scanning a phosphor screen using a Storm Phosphorimager (Molecular Dynamics). The 15% gel shows the results of ligations using octa-, nona- and decanucleotide libraries, labelled 8mer, 9mer and 10mer in the figure. Control reactions are in sets of three, one for each library, from left to right, without DNA ligase, M13mp18 ssDNA and library. Ligation rate is measured by the extent of the ladder. The image depicts the results of varying the concentration of each library. For each of the libraries, from left to right, the following concentrations were used: 8mer library, 8, 15, 30, 60, 90 and 120 fmol; 9mer library, 2, 4, 8, 15, 30 and 60 fmol; 10mer library, 2, 4 and 8 fmol. Each concentration represents that of each of the individual oligonucleotide species in the respective library. The arrow labelled 1 indicates the ³²P-labelled M13P directing primer. The arrow labelled 2 represents the first oligonucleotide ligated from each library. (B) The 2D graphic depicts the polymers analysed in (A). Each band intensity is expressed as a percentage of all the bands that represent ligated oligonucleotides.

Figure 3

Comparison of the rates of ligation of octanucleotides for four DNA ligases. (A) This is an image produced from radiolabelled ligation reactions after electrophoresis through a 15% polyacrylamide gel (see Materials and Methods). The left hand side of the gel depicts the control reactions lacking, from left to right, ligase, ssDNA template and octanucleotide library. The rate of the ligation reaction is determined by the length of the ladder and the intensity of each of the bands. This experiment used a complete octanucleotide library as substrate for each reaction (see text for details). The image depicts the results of varying the amount of each ligase, from left to right: Tth, 0.1, 0.3, 0.6, 1.3, 2.6, 5.1 and 6.3 pmol; Ts, 0.1, 0.3, 0.7, 1.3, 2.7, 5.4 and 8.0 pmol; Rm, 0.1, 0.2, 0.4, 0.8, 1.6, 3.1 and 4.6 pmol; Taq, 40.0 fmol and 0.1, 0.2, 0.4, 0.8 and 1.5 pmol. (B) The 2D graphic represents the polymers from (A) that were analysed. The intensity of each of the bands is expressed as a percentage of all the bands. Only the bands corresponding to oligonucleotide ligations were analysed.