Hi everyone! I am using a Q5 high fidelity DNA polymerase to amplify a 6.7 kb plasmid. The PCR with Q5 went well with longer primers (~55 bases) at 70-72 degree C. However, I keep failing in the PCR with shorter primers (~20-30 bases). The melting temperature of the shorter primers range from 60-65 degree C. To calculate the annealing temp, I put the primer sequence and my primer concentration (500 nM, working concentration in the 25 µL PCR reaction) in the NEB Q5 TM calculator, but the annealing temp it gave me is always at 72 deg C, but the PCR kept failing at 72 C. I also used gradient PCR to look for optimal annealing temperature from 60-72C, but none of them worked... I know that Q5 is different from Taq, and its annealing temperature is usually higher. Does anyone have any clue with my PCR with shorter primers keep failing? Should i design longer primers instead? Thank you
It may not be the primers at fault. The sequence may be GC rich so using 1M betaine or up to 8%dmso may work. Iwould try a temperature gradient of 55 to 70c and see what works in real practice
I am not an expert in this field, but I am very interested and have researched to find an answer. Could you please review the response below to see if it is correct?
The issue you are experiencing with the Q5 high-fidelity DNA polymerase and shorter primers can be attributed to several factors related to primer design, annealing temperatures, and the specific properties of the Q5 enzyme.
Firstly, it is important to recognize that Q5 DNA polymerase is known for its high fidelity and proofreading activity, which makes it more stringent in terms of primer-template mismatches compared to Taq polymerase [1][2]. This stringency can cause difficulties when using shorter primers that may not bind as stably to the template DNA. The longer primers you initially used (around 55 bases) likely provided more stable binding, even at higher annealing temperatures (70-72°C), which facilitated successful amplification.
However, when using shorter primers (20-30 bases), the melting temperature (Tm) is generally lower, and the binding is less stable. The NEB Q5 Tm calculator provides an annealing temperature of 72°C, which might be too high for these shorter primers, leading to insufficient primer-template hybridization and thus failed PCR reactions.
Given this context, here are a few suggestions to troubleshoot and improve your PCR protocol:
Lower the Annealing Temperature: Despite the NEB Tm calculator suggesting 72°C, it might be beneficial to conduct a more refined gradient PCR with a narrower range, focusing on lower temperatures. Try a gradient from 55°C to 65°C to see if there is an optimal temperature within this range where the shorter primers can effectively anneal.
Optimize Primer Design: If possible, redesign your primers to increase their length slightly. Even an increase to 30-35 bases can provide more stable binding without significantly altering the specificity of your primers. Additionally, ensure the primers have a balanced GC content (40-60%) to maintain a reasonable Tm.
Adjust Reaction Conditions: Modifying the concentration of MgCl2 and dNTPs can sometimes improve the efficiency of PCR with high-fidelity enzymes like Q5. Ensure that the MgCl2 concentration is not in large excess over the dNTP concentration to avoid destabilizing the primer-template complex [1].
Hot Start PCR: Although Q5 polymerase is less prone to non-specific amplification due to its high fidelity, employing a hot start PCR protocol might help. This approach can prevent primer-dimer formation and non-specific binding at lower temperatures.
Verify Primer Quality: Ensure that the primers are free from any secondary structures like hairpins or dimers, which can reduce the efficiency of the PCR. Tools like Primer3 or OligoAnalyzer can help in checking for these issues.
In summary, the challenges you are facing with shorter primers when using Q5 DNA polymerase are likely due to the stringent binding requirements and higher annealing temperature recommendations by the NEB calculator. By lowering the annealing temperature, slightly increasing primer length, and optimizing reaction conditions, you may achieve successful PCR amplification.
References:
[1] Eckert, K., & Kunkel, T. (1991). DNA polymerase fidelity and the polymerase chain reaction.. PCR methods and applications, 1 1, 17-24 .
[2] Fujiwara, H., Fujiwara, K., & Hashimoto, K. (1995). PCR with deoxyinosine-containing primers using DNA polymerases with proofreading activity.. PCR methods and applications, 4 4, 239-40 .
I received some assistance from tlooto.com for this response.
Hi Park Sowon Thank you for your help! I have tried gradient annealing temp from 53-72 deg C, but no temp worked for these shorter primers. I used 98 deg C as the hot start in all the Q5 PCR. After a series of troubleshooting, I was wondering if the failure was due to the low melting temp of these primers that prevented them from binding to the template when using Q5, so I re-designed the primers to 40 bases, with each melting temp around 70 deg C. I would keep you updated about my progress! Your reasoning made a lot of sense. Thank you so much!
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