What is touchdown PCR (TD-PCR) and when would I need to use it?
What is touchdown PCR (TD-PCR) and when would I need to use it?
During the PCR denaturation step, all DNA molecules will become single stranded. When the temperature decreases for annealing, three types of duplexes can be formed:
-Homoduplexes—annealing of complementary strands
-Heteroduplexes—cross-hybridization of homologous sequences that may have partial homology
-Duplexes between primers and template
To achieve higher specificity, heteroduplex formation should be minimized by increasing stringency (i.e., increasing the temperature) during the initial PCR cycles. Touchdown PCR increases specificity by using reaction conditions that gradually reduce the annealing temperature. The initial annealing temperature is set to several degrees above the estimated Tm of the primers. In subsequent cycles, the annealing temperature is slowly decreased until it reaches the calculated annealing temperature of the primers (Don 1991). By using a higher annealing temperature in the initial PCR cycles, touchdown PCR favors accumulation of amplicons for sequences with the highest primer-template complementarity, thereby enriching for the most specific amplicons. Transitioning to a lower temperature during subsequent cycles reduces stringency, improving priming conditions with the already enriched, desired template. We recommend performing an initial 5–10 cycles with the higher annealing temperature, and then gradually decreasing the temperature until the optimal annealing temperature, or ""touchdown temperature,"" is reached. For example, if the Tm of your primers is 68°C, the recommended TD-PCR conditions for the annealing temperature are:
-5 cycles at 72°C, then
-5 cycles at 70°C, then
->25 cycles at 68°C
References
Don, R. H., et al. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008 (1991).