Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction

We report a method for optimizing the specificity of product formation in the polymerase chain reaction (PCR). This technique is based on the use of dimethyl sulfoxide (DMSO) and takes into account primer Tm. The reduction in Tm by DMSO is directly correlated with renaturation temperature such that a DMSO gradient reflects a temperature gradient. We use this relationship to show that optimum product formation usually occurs at or within several degrees of the midpoint Tm of a given primer pair. We illustrate these correlations using three examples deriving PCR products from a human cDNA library, representing the casein kinase II alpha and beta subunits as well as the 5' untranslated region for the beta subunit. By following product formation as a function of renaturation temperature, we postulate rules for cycle design based on primer Tm. Implications for the use of degenerate primers are discussed.