The PCR enhancer increase the yield and the accuracy of the PCR result. The PCR reaction buffer enriched with different components such as Tris, EDTA, MgCl2, KCl, Formamide, DMSO, TritonX100, Nonidet P40, twin20, 7-deaza-2′-deoxyguanosine 5′-triphosphate and bovine serum albumin. These are the common PCR enhancers used into the PCR reaction.
“The PCR reaction buffer is the composition of different PCR enhancers that increase the efficiency and the specificity of the PCR reaction”.
Non-specific binding, primer- dimer formation, unwanted amplification, no amplification, low amplification and more than one amplification are some of the major problems occurring during the PCR reaction.
Long primers, high GC content in template DNA, unpurified template, PCR conditions and concentration of the chemicals used into the PCR reaction are the major reason for PCR failure.
In this article, the major focus will be on the different components used in the PCR reaction buffer.
If you want to master the PCR technique, you should learn how to use MgCl2 differently in different reactions. The MgCl2 is a major PCR enhancer present into the Taq DNA polymerase buffer.
The enzyme Taq required Mg2+ cofactor to start the catalytic reaction. This would facilitate by the addition of MgCl2. 0.5mM to 5.0mM MgCl2 can be utilised in PCR reaction. However, the concentration of the MgCl2 is depended on the condition of PCR.
Further, the Mg2+ of the MgCl2 binds to the PO3- (phosphate backbone of DNA) and increases the melting temperature of the reaction. This will facilitate more time for the primer to bind at its specific location.
Higher amplification can also be achieved with the use of MgCl2. Although, too much MgCl2 decreases the specificity of the reaction and increases the non-specific bindings. How? For that read the article: the role of MgCl2 in PCR reaction.
Furthermore, a higher concentration of MgCl2 decreases the rate of denaturation of template DNA by increasing the melting temperature.
This article contains all the information of MgCl2 and its role in PCR, how to use it, composition and limitations. Role of Mgcl2 in PCR reaction.
It facilitates the elongation of the Primer DNA complex. The K+ ion of the KCl binds to the DNA backbone (same as Mg2+). This binding decreases the electrostatic repulsion between two DNA strands and facilitates the elongation preferentially.
Increased concentration of KCl increases the power of denaturation especially in shorter DNAs (up to 1000bp PCR fragments). still, disappointing for the longer DNA.
In long-range PCR lower concentration of KCl is prudent. The final reaction concentration of the KCl in PCR reaction is between 25mM to 100mM.
Tips: For PCR of 2kb to 10kb DNA fragment use KCl below 25mM (you can also avoid it).
Some of the interesting articles:
DMSO is a major game changer in the PCR reaction with high GC content. To amplify the DNA with high GC content is quite difficult. The chance of secondary structure or hairpin formation is higher in high GC rich DNA.
It has great power of disrupting the hydrogen bond between the bases which increases the denaturation rate of high GC rich template.
DMSO prevents the hairpin formation by interrupting between two DNA strand reannealing. Also, it increases the binding specificity of primer to the template DNA.
Majorly, DMSO can be preferred into the PCR with GC content more than 55%. Use 4% to 10% DMSO into the PCR reaction buffer. Generally, 5% DMSO concentration is advisable and achievable.
Read an in-depth explanation of the role of DMSO in PCR reaction here: Role of DMSO in PCR reaction.
Glycerol is another reagent in the family of chemicals which reduce the secondary structure formation. The glycerol is impactful and works efficiently because it does not have any side effect in PCR reaction. 6% glycerol is recommended.
The formamide is less known for its role in PCR enhancement. It is the best alternative for DMSO. formamide increases the PCR efficiency of the GC rich region too. A concentration between 1% to 10% is preferable.
Use the formamide-based on the content of the GC into the DNA. It increases the primer binding efficiency.
Once the DNA template denatured, no hydrogen bonds are present between bases. In that case, especially, in long DNA template, the DNA becomes bound with each other and forms the secondary structure or hairpin structure.
The secondary structure or the hairpin stabilised the ssDNA and hinders in elongation of the DNA. 7-deaza-2′-deoxyguanosine 5′-triphosphate is another chemical that prevents the secondary structure formation into the denatured single-stranded DNA.
It prevents the false results into the PCR. The approximate concentration of dc7 GTP is ~40µM.
Betaine, N,N,N-trimethylglycine is a minor PCR additive that prevents the secondary structure formation especially, into the high GC rich region. It increases the yield in the high GC rich PCR reaction.
Nonetheless, the exact role of betaine in amplification of high GC rich PCR is controversial and not understand properly.
Notably, along with DMSO, betaine increases the specificity of high GC rich long-range PCR reaction. The ideal concentration of betaine in PCR should be between 0.5M to 3.0M.
Bovine serum albumin:
DNA extracted from the samples such as marine water, soil, fresh water or faeces samples contains a high level of PCR inhibitors such as fulvic acid, humic acid, tannic acid FeCl3 and other.
These inhibitors can hinder in the elongation of the newly grown DNA strand. BSA, a natural protein can prevent the PCR reaction from such types of inhibitors.
The viscous nature of BSA prevents sticking of PCR reagents on to the wall of the PCR tube. The concentration of bovine serum albumin should be between 300 ng/μl to 600 ng/μl into the PCR reaction.
T4 gene 32 protein:
This protein also prevents the PCR reactions from the inhibitors, however, which types of inhibitor it blocks still not known well. The ideal concentration of T4 gene 32 protein should be ~100 ng/μl.
Non- ionic detergents:
Detergents are normally used into the DNA extraction. Common detergents such as Triton X100, Nnonidet P40, Tween20 and SDS is majorly involved in the cell lysis.
However, apart from SDS and CTAB, other non-ionic detergents as like Triton X100, Nonidet P40 and Tween20 are great PCR enhancers.
Using these detergents in PCR reaction buffer can greatly increase the yield and efficiency of the PCR reaction.
The detergents can suppress the activity of other contaminants such as SDS, phenol and other DNA extraction buffer traces. It neutralises the inhibitory effects of contaminants.
However, excess concentration of the non-ionic detergents can inhibit the PCR reaction. 1% to 2% of non-ionic detergents are sufficient and can be used in the PCR reaction buffer.
It can also increase the efficiency of GC rich PCR reaction. However, it is less known for its role in PCR reaction buffer, still you can you use it 100mM in reaction buffer.
Additionally, it increases the specificity of the primer and DNA hybridization and does not allow any non-specific bindings.
Polyethylene glycol is less known for its role in PCR reaction buffer. PEG is the excellent enhancer of the PCR reaction. It works differently than other chemicals. It prevents the re-annealing of denatured single-stranded DNA by binding with the ssDNA.
Read more on DNA replication:
- DNA Replication class 1: General process of DNA replication
- Prokaryotic DNA replication: Replication class 2
Interestingly, it blocks the unbounded primers and prevents dimer formation during the PCR reaction. For efficient PCR reaction buffer preparation use PEG between the concentration of 5% to 15%.
It is viscous in nature which prevents the bindings of Taq DNA polymerase and other PCR reaction reagents to the wall of the PCR tube. By doing this, it provides more surface area to the reaction between different chemicals.
Also, it prevents excess evaporation during the extreme heating in the PCR. The function of gelatine is somewhat similar to the BSA. Still, we can use it between the concentration of 0.01% to 0.1%.
Bonus Tips: while working on the samples of ancients tissues or ancient DNA and DNA samples having more melanin, use Bovine serum albumin. The concentration of BSA is given above, this will increase the possibilities of PCR results.
The above-enlisted chemicals are PCR enhancers which can be used into the PCR reaction buffer, yet, some other chemicals which are as important as the PCR enhancers. These chemicals are used to maintain the pH and the stability of the reaction.
Tris and EDTA are two important ingredients serve the purpose of stabilising the reaction. The DNA is pH sensitive, constant pH environment between 7.8 to 8.5 is required for DNA to work efficiently which stabilise the reaction.
Tris-HCl with pH 8.0 maintains the stability of the reaction and constant pH throughout the PCR reaction. This prevents the degradation of the DNA molecule.
EDTA is a chelating agent and blocks the activity of metal ion, especially mg2+. In this condition, if Mg2+ is not available then how Taq DNA polymerase can work?
The question is valid because if we use EDTA, it definitely suppresses the activity of Mg2+, but it can also enhance the reaction.
Using EDTA into PCR will chelate the excess unused Mg2+ ions that might hinder into the PCR, also, it chelates other ions presents into the PCR reaction mixture. Therefore, if we use EDTA in a lesser proportion of MgCl2, it will enhance the PCR reaction.
Tris and EDTA are two major components that are required in each and every step of performing any experiments with DNA.
We had covered a whole article on the role of Tris and EDTA, read the article here: Importance of Tris-EDTA (TE) buffer in DNA extraction.
Summary of the PCR reaction buffer:
GC rich template DNA: use 7-deaza-2′-deoxyguanosine 5’-triphosphate, betaine, DMSO, formamide, glycerol and Tetramethylammonium chloride.
Prevent secondary structure formation: formamide, DMSO, glycerol, betaine and 7-deaza-2′-deoxyguanosine 5’-triphosphate
Increase primer binding efficiency: MgCl2, KCl and Tetramethylammonium chloride
Increase Tm: Tetramethylammonium chloride
Decrease Tm: DMSO
Enhance Taq polymerase activity: MgCl2, polyethyline glycol and gelatine.
My ultimate guide for optimising PCR reaction buffer:
The condition of any PCR reaction varies from lab to lab and person to person. The protocol that I design, might not work properly for you and it is very often in molecular biology.
Though we have given concentration for all the different components which are used in the PCR reaction buffer preparation, you have to optimise or standardise your own protocol into the lab.
We are explaining it here. Let’s take the example of MgCl2. Our aim is to optimise the MgCl2 in our reaction.
Six different temperature gradients are there in our gradient-conventional PCR machine each with the minimum temperature difference of 0.5°C. First, we will find out the annealing temperature for our PCR reaction,
Mix all reagents as per the protocol, suppose our annealing temperature (by primer 3) is 61°C. Now design 6 different tubes and put is in the PCR machine. Set the temperature ranging from 59°C to 63°C (59°C, 60°C, 61°C, 61.5°C, 62°C, 63°C). The condition is shown int to the figure below,
After agarose gel electrophoresis, suppose we get some amplification at the temperature 62°C. Now we have to enhance that PCR reaction results.
Prepare another set of reaction as per the protocol. Add MgCl2 0.5mM to 5 mM.
Tube 1- 0.5mM
Tube 2- 1.0mM
Tube 3- 2.0mM
Tube 4- 3.0mM
Tube 5- 4.0mM
Tube 6- 5.0mM
Place it into the PCR machine at 62°C temperature analyse the results on 2% agarose gel. Or you can follow another method which is tedious but quite accurate, the method is shown in the figure below,
Finally, we are giving you our recipe of the PCR reaction buffer, what components we are using in preparing the PCR buffer.
Preparation of 10X PCR reaction buffer:
100mM (pH 8.0)
Ammonium sulphate (NH4)2SO4
This recipe of the PCR reaction buffer is for 55% to 60% of GC content into the DNA which we are using in our lab. You can use this buffer directly or you can use one of the following chemicals as per your PCR need.
Read more on PCR:
- A Complete Guide of the Polymerase Chain Reaction
- The Function of dNTPs in PCR reaction
- Role of DMSO in PCR: DMSO a PCR enhancer
- Function of taq DNA polymerase in PCR
- PCR primer design guidelines
- Role of MgCl2 in PCR reaction
Experience and expertise are required to play with the different components used in the PCR reaction buffer. Nonetheless, by doing the systematic and organise PCR protocol standardization experiments anyone can achieve expertise and achieve excellent results into the PCR.
Remember, never satisfied with your results because a combination of chemicals what you are using today that might not work in future.