216 Maunders2.2.3. Temperature
Rates of reaction are very temperature-sensitive, the optimum depend-
ing on the lengths of the oligonucleotides being joined, and being
greater than the T m of the substrate. E. coli DNA ligase has an optimum
temperature of 10-15°C for ligating cohesive ends (27).
Another bacterial ligase from the thermophilic bacterium Thermus
thermophilus (28,29) is becoming widely used as further applications
of the polymerase chain reaction are developed (30). The enzyme is
similar to E. coli DNA ligase in size, pH optimum, cofactor and cation
requirement, and response to activators. However, its temperature
optimum is much higher, being 24-37°C for ligating cohesive termini,
and 65-72°C for closing nicks (within a range of 15-85°C), which
may result in more widespread applications of the technique of ligation.
T4 DNA ligase has an optimum temperature for ligating cohesive
ends of 4°C (3I) and for sealing nicks of 37°C (32). The optimal
temperature for blunt-end ligation is 25°C for 16 mers or longer, smaller
molecules requiring lower temperatures consistent with their decreased
melting temperatures.
2.2.4. pH
Optimal pH for ligation varies to some extent with the reaction
catalyzed and the buffer employed. E. coli DNA ligase has a pH op-
timum for joining DNA strands of 7.5-8.0 in Tris-HC1 buffers and of
8.0 in sodium phosphate (33). For the phosphate exchange reaction,
the optimal pH is 6.5 in potassium phosphate, the rate of reaction
falling to 50% at pH 5.6 and 7.5. This reaction rate falls to 20% in Tris-
HC1 buffer at pH 8.0.
T4 DNA ligase has a similar pH optimum range for joining DNA,
the reaction rates falling to 40% at pH 6.9 and 65% at pH 8.3. The pH
optimum for the exchange reaction is also similar to that for E. coli
DNA ligase. For the sealing of nicks, the optimal pH range is 7.2-7.8
in Tris-HC1, with the rates dropping to 46% at pH 6.9 and to 65% at pH
8.5 (17,33).
2.2.5. Cations
DNA ligases require a divalent cation, with Mg 2÷ being the most com-
monly utilized. The E. coli enzyme requires Mg 2÷ at an optimum concen-
tration of 1-3 mM. Low concentrations of Mn 2+ can substitute, and in the