PCR inhibitors are chemical or physical obstacles which block amplification and fails the PCR reaction. Contaminants in the sample, DNA or in buffer solutions used into the PCR reaction are some of the common PCR inhibitors.
PCR is the temperature-dependent cyclic enzymatic reaction in which the DNA is amplified. Our website is full of so many articles and explanation on the PCR, read it. We are not explaining here about the PCR.
Aside from the clinical diagnosis, PCR is used in the food industries, forensic analysis and environmental studies as well.
Several chemicals present in the PCR reaction buffer increases the efficiency and the specificity of the PCR reaction. Some of them are MgCl2, DMSO, gelatine, BSA, KCl and some other salts.
However, some other chemicals and physical agents hinder in the PCR reaction. In this article, we will explain how different PCR inhibitors inhibits the PCR reaction and how can we overcome this problem.
Key Topics:
What is PCR inhibitor?
An inhibitor inhibits or stops any biological reaction, the entire process is known as inhibition. It might be physical agents, chemical agents or anything.
Enzymatic inhibitors and reaction inhibitors are the two most common types of inhibitors in any biological reaction. As we all known that any biological reaction is governed by an enzyme hence if the enzyme is inhibited, ultimately the reaction is inhibited.
Now coming to the point,
The PCR is also an enzymatic chemical reaction in which the DNA is amplified. Here if the activity of the enzyme Taq DNA polymerase is inhibited consequently the amplification process is stopped.
Also, if any of the chemical contaminants are present into the PCR template DNA, the reaction is inhibited. Furthermore, if the reaction is not performed well “with good technical hands”, the reaction fails and amplification is not achieved.
DNA extraction chemicals such as CTAB, SDS, NaCl, phenol-chloroform other detergents and KCl are some of the common types of PCR inhibitors observed.
The Taq DNA polymerase remains inactive if MgCl2 in very low into the PCR reaction buffer hence the reaction is inhibited.
Before understanding the mechanism of how inhibitors stop the PCR reaction, we have to learn how does amplification fails in PCR.
How does amplification fail?
There are several reasons in which PCR inhibitors are involved and in some other reasons, PCR inhibitors are not involved.
For example, if any of the PCR reagents are not added in the appropriate concentration, the reaction fails. Preliminarily, if the DNA template is not added into the PCR reaction the reaction is failed and amplification is not achieved.
Likewise, if any of the reagents from PCR primers, dNTPs, MgCl2 and PCR reaction buffer are not added into the reaction. The amplification is not obtained.
In another reason, if one of the chemicals is not working properly or degraded, the reaction fails. For example, if primers are not well maintained at -20°C temperature, it fails to amplify the DNA.
If the annealing temperature is not appropriate or too high, amplification cannot be achieved.
If the PCR cycles and the time for each step are not properly chosen, it will result in PCR reaction failure.
These are some of the common causes of PCR failure, apart from these there are N numbers of a reason for not getting the amplification.
Now let’s discuss some of the common PCR inhibitors.
Read more:
What are the different types of PCR inhibitors?
Sample collection, DNA extraction, DNA purification and sample preparation are some of the steps in the PCR.
different types of PCR inhibitors originated from different steps.
During the sample collection,
The cell contains different proteins and lipids such as polysaccharides, polyphenol compounds, cell wall proteins, Haem part of haemoglobin, lipids, hormones and other molecules greatly influence the PCR reaction and fails the amplification.
If traces of any of the compounds remains into the DNA, it will hinder into the reaction.
During the DNA extraction,
We are using different organic and inorganic chemicals. Depending upon the types of chemicals it is categorised into organic DNA extraction methods or inorganic DNA extraction methods.
The DNA extraction is performed to separate nucleic acid from cell organelles and to remove other cell compounds like proteins and lipid.
For doing that we are using different chemicals such as SDS, phenol, chloroform, isoamyl alcohol and CTAB etc. However, any part of these compounds, if remains into the nucleic acid it will bind to DNA and hurdle the PCR amplification.
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During the sample preparation,
For removing all the contaminants from the DNA we have to purify it with the help of the alcohol. Alcohol removes traces of all the chemicals and other impurities and reduces the chance of reaction failure. Nucleic acid purification is a crucial step in DNA extraction.
Nonetheless, alcohol itself is an inhibitor of PCR. If the DNA is not dried well after the purification, the alcohol remains into the DNA hurdles into DNA dissolving.
Yet, alcohol is an important ingredient in DNA extraction, it precipitates the DNA. To know more on the mechanism of precipitation, read our article: Role of alcohol in DNA extraction
Additionally, PCR inhibitors are present in different biological materials. Take a look at the chart below,
Effect of some of the common PCR inhibitors on PCR amplification:
The plant polysaccharides reduce the precipitation capacity of the nucleic acid.
The presence of Urea and proteas inhibits the enzyme activity whereas some of the detergents are responsible for the degradation of the Taq DNA polymerase enzyme.
The specificity of the primers is decreased in the presence of the metal ions or in the presence of the higher concentration of metal ions.
The chemical properties of nucleic acid altered in the presence of the polyphenolic compounds, polysaccharides, proteins, detergents, salts and other PCR additives by cross-linking with DNA molecule.
Interestingly, the polyphenolic compounds present into the plant co-precipitated along with the nucleic acid and reduce the purity of the nucleic acids.
The rate of the DNA elongation is reduced by the presence of the antiviral compounds such as acyclovir.
The Taq DNA polymerase required a cofactor for their optimum activity, however, the activity of the Taq DNA polymerase is reduced in the absence of the metal ions such as Mg2+.
Also, some of the metal ions such as calcium competes with the Mg+ and reduces its binding capacity to the Taq DNA polymerase.
EDTA, the chelating agents is also one of the reasons for the inactivation of Taq.
Collagens, heparins, immunoglobulins (especially the IgG), Haemoglobin, proteinase, nuclease, Urea, bile and salt are some of the common types of PCR inhibitors originated from the biological tissues.
Apart from all these inhibitors one major inhibitor of the PCR reaction is not from the list. Plasticware and glassware are also involved in the PCR inhibition.
Powder and chemicals present on the gloves and other plasticware, unautoclaved plastic wares and impurities present into the plastic hinder into the amplification. The nuclease present in PCR tubes, tips and glasswares prevent amplification by degrading the nucleic acid.
Bonus tips:
If amplification, primer-dimer or non-specific bindings are not present into the results, possibly, one of the ingredients is absent into the sample. Inhibitors cannot be involved in the reaction.
If primer-dimers are present but no bands observed into the results, PCR inhibitors might be involved in the amplification process.
If primer-dimers and non-specific bands are present into the results but the desired amplicon is absent, indicates strongly that some of the inhibitors are present or other template DNA (as an inhibitor) competes the target template DNA.
How to reduce PCR inhibitors from PCR reaction?
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- Maintain proper sterile condition during the sample processing, DNA extraction and PCR reaction preparation.
- Wear gloves, mouth cap and lab coat to reduce the chance of nuclease contamination into the sample.
- Use a proper purification method, use a spin column DNA purification method which removes almost all the compounds bound to the DNA. I recommended using “ready to use kit” for DNA extraction.
- Use PCR additives to minimize the activity of the PCR inhibitors.
- Use DD/W for dissolving DNA instead of the TE buffer.
- Furthermore, to find out the presence or absence of PCR inhibitors, use controls into the reaction or perform spectroscopic analysis. Pure DNA and Pure RNA has OD nearly 1.8 and 2.0 respectively.
- If the ration of the 260/280 will be more or less than the ideal range indicates the presence of the inhibitor into the reaction.
“Other DNA present in the sample also inhibits the PCR reaction as it compites with the target template DNA.”
Other resources related to this topic:
Importance of TE buffer:
-Tris, EDTA, preparation of 10X TE and importance of TE buffer.
Different types of DNA extraction methods:
-History of DNA extraction, how to extract DNA and different methods of DNA Extraction
Conclusion:
The polymerase chain reaction is one of the important tools for scientist into the genomic research, yet, PCR inhibitors are the major limitation of it, especially in forensic science where the sample is a piece of crucial evidence in the identification of the suspect. Doing sample processing and DNA extraction very preciously is the very first step to achieve ideal amplification in the PCR reaction.
