Polymerase chain reaction is a highly sensitive and specific technique, however, non-specific reactions can also occur here. How can I troubleshoot such problems? And how I can avoid from these reactions?
This is a vast topic. A very good trouble-shooting guide is included in the QIAGEN DNA polymerase manuals. I will send you one in the attachment.
Primer design is a very critical step and much can be lost or gained by bad or good primers. Optimization of the PCR cycling is also a very powerful way to improve your protocol. It may also be worth trying different DNA polymerase brands. Enzymes from different manufacturers do not perform in the same way. Use aerosol-tight pipette tips, if available.
Experimental controls are very important. Besides your samples of interest, you should as well include one reaction with a positive control DNA. This can be a related gene from a different organism. And you absolutely must include a negative control reaction, where you do not add any DNA. If you see any amplification in the negative control, discard the whole reaction series, as the results cannot be trusted. You will then optimize your protocol until the negative control reaction remains clear. Store buffers, primers and dNTPs in small aliquots. This makes it easier to discard them if they are suspect of contamination.
It is also a good idea to use seperate lab equipment and even separate rooms to handle DNA preparation, PCR mastermix setup and electrophoresis/preparation of PCR products. Do not cross-use pipettes between these tasks. The best template for PCR is a stretch of DNA which has already been amplified by the method!
Some people put their PCR reaction products and corresponding waste into the autoclave for proper sterilzation. This is a very bad idea. PCR amplification products do not need to be sterilized, as all living cells have already been removed during the DNA exctraction procedure. Dispose them in the normal (non-criticial) waste instead. After autoclaving, the PCR amplification products will be spread in the autoclave and in the whole room as a very fine aerosol and you will never be able to clean-up the room and equipment again.
There are more points to be mentioned, but these are very important ones.
P.S.: I have once visited a very big lab and there I saw how far such precautions can go. That lab handled many thousands of samples every day in standard tests and it was important to find the very few positive cases each day. The staff from the pre-mastermix labs and staff from the labs handling the PCR amplification products were not even allowed to take lunch together and use the same restrooms. They really had separate lunch rooms for each staff, as the possibility of cross-contaminating the PCR reaction via the people and their clothing was considered too high a risk!
For that particular lab, this made sense indeed, as every contamination of the tests would have costed a great amount of money.
1. Use high purity DNA samples.
2. The primers used for PCR should be unique/specific to your target.
3. The setting of Ta (annealing temperature) should be proper (according to the Tm of your two primers)
4. Avoid sample contamination, such as properly use the Pipetman for adding different samples
5. Avoid distraction when you are preparing PCR samples
1. Too many cycles were used: Excessive cycling increases the opportunity for nonspecific amplification and errors. Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low.
2. Extension time was too long: Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1 min/kb.
3. Annealing time was too long: Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec.
4. Annealing temperature was too low: If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient.
5. Thermal cycler ramping speed is too slow: If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and sufficient time for nonspecific binding. If ramping speed is not set at the maximum speed for the cycler, increase to maximum ramp rate.
6. Calculated primer Tm was inaccurate: If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be incorrect. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature.
The main reasons of getting non specific amplification during PCR reaction are-
1. Use of primers that bind to other regions instead of the target gene leading to nonspecific amplification
2. Improper annealing as well as melting temperature that tends the primer to bind to different regions randomly
3.purity of sample (starting from isolation of RNA then cDNA preparation)
Hpope this information can help you
You can please follow the links below to obtain certain valuable informations:-
1. http://www.bio-rad.com/en-us/applications-technologies/pcr-troubleshooting
2. http://www.sigmaaldrich.com/technical-documents/protocols/biology/hot-start-dna-amplification.html
3. http://www.highveld.com/pcr/pcr-troubleshooting.html
4. https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide
Hello everyone,
May you can explain for me the reason why "Excessive extension time can allow nonspecific amplification" ?
Thank you so much.
..yes to increase annealing temp maybe an option (but, should be lower than primer Tm)
# long annealing time may predispose non-specific reactions....
If you do not have any problem with your primers I suggest to optimize annealing temperature using gradient pcr. As the annealing temperature increases, the specifity will increase and it is possible to get desired bands.
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