Specificity-Enhanced Hot-Start PCR: Addition of Double-Stranded DNA Fragments Adapted to the Annealing Temperature

A new method to produce hot-start conditions in PCR is described. Short double-stranded DNA fragments were found to inhibit the activity of DNA polymerases from Thermus aquaticus and Thermus flavus. This inhibition is not sequence specific, but exclusively dependent on the melting temperature of the fragments as shown by its correlation to their melting curves as measured. This property is exploited by adding fragments of the appropriate length to the PCR mixture during the reaction setup and thereby preventing the DNA polymerases from extending primers annealed nonspecifically at lower than the optimal temperature. By amplifying ten copies of phage λ DNA in the presence of 2 μg of nonspecific DNA, it is shown for three different primer pairs how the melting temperatures of the double-stranded DNA fragments have to be adapted to the cycle profiles to obtain predominantly specific products in the 0.5 μg range.