FIELD

This disclosure relates to the use of one or more proteins (e.g., globular proteins) having a low isoelectric point and/or a limited number (e.g., zero) of modifying groups in nucleic acid polymerization and/or amplification reactions such as polymerase chain reaction (PCR).

BACKGROUND

Many widely known recombinant nucleic acid (e.g., DNA, RNA) techniques involve polymerizing and/or amplifying DNA. One such example is the polymerase chain reaction (PCR). During PCR, the reaction cycles repeatedly between at least two temperatures, a low and a high temperature (e.g., 55° C. and 95° C.) in the presence of a thermostable DNA polymerase enzyme. The total period of time spent at the high temperature over the course of the reaction depends upon the total number of cycles, the duration of the high temperature step of each cycle, and the ramp speed (i.e., the rate at which the thermocycler changes from one temperature to another). Although the DNA polymerases used in PCR are highly thermostable, they tend to become inactive at high temperatures over time. Furthermore, these polymerases may also become inactive by being introduced into reaction mixture environments with sub-optimal concentrations of cofactors, or that have sub-optimal pH levels, or that include the presence of chemical or biological inhibitors.

One way of stabilizing an enzyme under such conditions is to add a stabilizing agent, such as a surfactant. Surfactants, such as detergents, are surface-active compounds that stabilize the interface between the active form of an enzyme and its liquid environment. For example, the activity of Taq DNA polymerase has been stabilized by the addition of nonionic detergents, such as NP-40 or Tween® 20 (Bachmann, et al.Nuc. Acids Res.18(5): 1309 (1990)). In some applications, however, detergent-stabilized (e.g., Tween® 20) DNA polymerases have low efficiencies of amplification or lead to the amplification of non-specific products. There is a need, therefore, for reagents and methods that improve the efficiency and/or stability of thermostable DNA polymerases in solution during polymerization and/or amplification reactions.

