DNA is a dynamic molecule with a structure that is stabilized by a large number of weak interactions. The stability of the DNA double-helix depends on a variety of factors, including DNA sequence, pH, ionic strength, solvents and temperature. In particular, as the temperature is increased, the weak interactions are sequentially disrupted, first resulting in localized denaturation of the terminal and selected internal sequences, and finally resulting in complete separation of DNA strands. The degree to which this destabilization is desired or tolerated depends on the application.
For example, cloning procedures, such as end-polishing are maximized when the termini are stabilized, suggesting use of a polymerase derived from a mesophilic organism. Second strand cDNA synthesis and nick translation are other applications that traditionally use mesophilic enzymes. Such enzymes are maximally active at temperatures of 25–40°C and retain significant activity at lower temperatures as well. Generally, they can be heat-inactivated and work in the same buffers used by restriction endonucleases and ligases, obviating the need for intermediate DNA purification.
A variety of other molecular biology applications, such as PCR, require high temperatures to denature the DNA prior to primer annealing or during polymerization to reduce secondary structure, thus reducing polymerase pausing. Archaeal DNA polymerases, such as Vent® (NEB #M0254) and 9°Nm™ (NEB #M0260) are derived from hyperthermophiles and are extremely resistant to heat inactivation, even at 100°C, and display maximal polymerase activity at 75–85°C. Bacterial thermophiles have yielded enzymes such as Taq DNA polymerase, which is active at similar temperatures, but is not quite as stable at 95°C, as some of its archaeal counterparts.
Vent® is a registered trademark of New England Biolabs, Inc.
9°Nm™ is a trademark of New England Biolabs, inc.