Highly processive DNA polymerases are beneficial for GC-rich PCR, because of their strong binding to the templates during primer extension ( Figure 6B). These reagents often lower the primer T m, so the annealing temperature should be adjusted accordingly. To overcome strong GC interactions, the most common approach relies on PCR additives or co-solvents such as DMSO to help DNA denature ( Figure 6A). To amplify GC-rich targets, the double-stranded template must be separated for the primers to bind and DNA polymerase to read through the sequence. Thus, GC-rich sequences can cause DNA polymerases to “stutter” along templates and interrupt DNA synthesis. GC-rich sequences can also be involved in secondary structures. In this manner, desired PCR products are selectively increased with little or no amplification of nonspecific targets over the course of PCR ( Figure 2).ĭNA templates containing high GC content (>65%) can be difficult to amplify because of the stronger hydrogen bonds between G and C bases. Once the annealing temperature reaches, or “touches down”, at the optimal temperature (usually 3–5☌ lower than the lowest primer T m), it is maintained throughout the remaining cycles for primer annealing. To overcome this challenge, the annealing temperature is often decreased 1☌ at every cycle of the initial few cycles to produce a sufficient yield of the desired amplicon. While preventing primer-dimers and nonspecific primer binding, the higher annealing temperatures may result in lower PCR yield due to increased dissociation of primers from their intended target. As such, higher annealing temperatures reduce nonspecific PCR products and promote specific amplification at the start of PCR (learn more about PCR annealing step). Higher temperatures help destabilize the formation of primer-dimers and nonspecific primer-template complexes, thus minimizing undesirable amplification. ![]() In touchdown PCR, the annealing temperature of the first few cycles is set to be a few degrees higher than the highest melting temperature (T m) of the primers. Another approach to promoting specificity is to modify the PCR cycling parameters.
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