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

AUTOR(ES)

Housby, J. Nicholas

FONTE

Oxford University Press

RESUMO

We describe the characterisation of four thermostable 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 oligonucleotides initiated from a 17 base ‘primer’, using M13mp18 ssDNA as template. Polymers of ligation products were analysed by polyacrylamide gel electrophoresis. 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 ~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.

Documentos Relacionados

• DNA Ligases: Progress and Prospects*
• Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties
• A DnaB intein in Rhodothermus marinus: Indication of recent intein homing across remotely related organisms
• The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species.
• Creation of genetic information by DNA polymerase of the thermophilic bacterium Thermus thermophilus.