Combined Hot Start and Touchdown PCR Protocols Optimize Amplification of Difficult Targets

摘要

Blends of thermostable DNA polymerases such as Roche Molecular Biochemicals' Expand High Fidelity DNA Polymerase are an excellent choice to amplify target DNA sequences with high yield and accuracy. Occasionally, however, certain DNA targets are difficult to amplify: these reactions may fail or may contain non-specific products. Addition of the enzyme at temperatures above the oligonucleotide primer annealing temperature, known as "hot start" PCR, improves the amplification specificity because mis-annealed primers, which occur at lower temperatures, are not extended to generate non-target products (1). A second approach, "touchdown" PCR, assumes that the melting temperature of a correct primer/template pair is higher than that of an incorrect mismatched primer/template pair (2). Touchdown PCR protocols set annealing temperatures above the anticipated melting temperature of a primer/template pair for the first few cycles and then decrease the annealing temperature of subsequent cycles stepwise to allow correct primer/template pairs to form. This results in the amplification of a desired product before spurious priming of non-specific products can occur. We describe how hot start and touchdown PCR approaches can be combined to successfully amplify difficult target sequences with high yield and specificity using Expand High Fidelity DNA Polymerase.