Gradient Thermal Cycler: A “Hidden Trick” to Reduce Nonspecific Amplification

Why Does PCR Always Produce a Bunch of Nonspecific Bands?

One of the most frustrating issues in PCR is when the electrophoresis results show a bunch of random nonspecific bands in addition to the target band. Sometimes, the primer dimers are even brighter than the target band. When this happens, don’t rush to blame the primers or the template. First, check whether you’ve used the gradient screening function of your gradient thermal cycler correctly.

Use a Gradient Thermal Cycler to Quickly Find the Optimal Annealing Temperature

The most common cause of nonspecific amplification is an annealing temperature that is too low. This allows the primers to bind to nonspecific sites on the template. In this case, rather than redesigning the primers, it’s better to first perform a temperature gradient screening using a gradient thermal cycler. The method is simple: based on the primer’s Tm value, set a temperature range with a 5–10°C fluctuation. For example, if the primer’s Tm is 60°C, set a gradient from 55°C to 65°C. Run 2–3 replicates at each temperature, including a negative control. After gel electrophoresis, identify the temperature at which the target band is the brightest and nonspecific bands are minimal—that is your optimal annealing temperature.

A “Hidden Trick” to Reduce Nonspecific Amplification

If nonspecific bands are still present after gradient screening, you can try the “Touchdown PCR” function on your gradient thermal cycler. This function uses a higher annealing temperature during the first few cycles. Then, the temperature is lowered by 0.5–1°C per cycle until it reaches the final annealing temperature you set. This allows the primers to preferentially bind to the perfectly matched target site. This significantly reduces the likelihood of nonspecific amplification. In many cases, samples that fail to produce clean bands with standard protocols can yield excellent results using a touchdown PCR protocol.

What to do when amplifying high-GC templates is difficult?

If your template has a particularly high GC content (over 60%), you can add 5%–10% DMSO or glycerol to the reaction mixture while setting the temperature gradient. Then, use the gradient thermal cycler to screen for higher annealing temperatures; this often resolves amplification difficulties with high-GC templates.

In fact, most PCR amplification issues can be resolved through condition optimization using a gradient thermal cycler.