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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Housby JN,Thorbjarnardóttir SH,Jónsson ZO,Southern EMdoi
10.1093/nar/28.3.e10keywords:
subject
Has Abstractpub_date
2000-02-01 00:00:00pages
E10issue
3eissn
0305-1048issn
1362-4962pii
gnd010journal_volume
28pub_type
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