“DMSO is a PCR enhancer increases yield and specificity in PCR.”
One of the types of PCR additive, DMSO, commonly used during the PCR reaction for optimizing it. Besides, several other PCR enhancer or additives are MgCl2, KCl, albumin, (NH4)2 SO4 and other salts.
Polymerase chain reaction-PCR is an in vitro process of DNA amplification. It is adapted in genetics and genomics research. In fact, PCR amplification is one of the prime steps in every reaction, almost.
Taq DNA polymerase, dNTPs, template DNA, PCR buffer, DNA primers and water are common ingredients for PCR. These are needed in every PCR reaction.
Although all ingredients are equally important in PCR, PCR buffer plays an important role in getting amplicons. MgCl2, KCl, albumin, (NH4)2 SO4 and DMSO are popular components of PCR reaction buffer. Not for all the templates, but for harder template DNA, PCR reaction buffer must be required.
Longer and high GC rich DNA are hard to amplify with basic PCR components and normal PCR protocol. the PCR additives help in achieving amplification for these templates.
In the present article, we will talk about the special player of PCR and a key ingredient of the PCR buffer- the DMSO. If you are very much interested in learning PCR, this article is definitely for you.
So please read the article till the end.
Structure of DMSO:
DMSO, Dimethyl sulfoxide is an organic solvent molecule.
As shown in the figure, it has the trigonal pyramidal symmetry. The unique solvent and melting properties of DMSO makes it unique above all organic compounds and hence it is applicable in molecular biology and as a Cryoprotectant in medicinal research. Several properties of DMSO are listed here,
Properties of DMSO
- It is a polar solvent and dissolves in both, polar and non-polar solutions.
- It has a higher melting and boiling point.
- The dielectric constant of DMSO is ~ 48. 9.
- The pKa value is 35.1
Interestingly, not everyone is familiar with the DMSO and how to use it in PCR because it is used only in some special type of PCR reactions. And as we said above, one of the special types of PCR reaction is an amplification of high GC rich template DNA.
GC content of templet DNA is one of the reasons for PCR failure. It results in either reaction failure or non-specific amplification. Thus results are non-conclusive.
The DNA is made up of A, T, G and C. Two hydrogen bond between A and T and three hydrogen bond between G and C are present.
The triple hydrogen bond between G and C makes them more stable during PCR. Therefore higher than normal temperature requires to amplify it. Their stability also induces secondary structure formation during the PCR as well.
In addition to this, it also increases the primer-dimer formation. In a simple language, we can say, we can’t get what we want!
DMSO is added in the PCR reaction to overcome this problem.
Role of DMSO in PCR:
DMSO makes GC rich DNA more heat-labile and reduces the Tm (melting temperature) of reaction.
Here, DMSO directly binds to the cytosine residue of the GC rich region and changes the conformation of cytosine which makes it more heat-labile.
Hypothetically, DMSO reduces the strength of the hydrogen bond between the major and the minor groove of DNA. The DNA structure becomes unstable which ultimately lowers the denaturation temperature.
DMSO even prevents secondary structure formation.
Due to the high GC content in DNA, the DNA creates the secondary structure or hairpin loop. Three hydrogen bond requires more energy to broke, the secondary structure is formed by binding of single-stranded DNA with each other, just like a hairpin loop.
The hairpin DNA can’t amplify properly during PCR and results in PCR failure.
DMSO binds to the DNA and prevents the reannealing of single-stranded DNA. It also facilitates the annealing of primer with a templet. Therefore, it increases the specificity and yield of PCR reaction.
Generally, the GC content of the template DNA for PCR is between 45% to 52%. If the GC content is higher than the desired range use 5% DMSO in PCR reaction. 4% to 10% DMSO concentration can be utilized to optimize the PCR reaction.
Furthermore, the concentration of the DMSO in PCR depends on the type of reaction, the GC content of DNA and the quality of DMSO.
Interestingly, besides increasing specificity, it changes the DNA topology as well.
Recent studies on Plasmid DNA confirms that DMSO releases DNA supercoiling. Here, DMSO greatly induces topoisomerase I activity. The topoisomerases are the class of DNA enzymes helps in relaxing DNA.
Therefore, it boosts the reaction specificity by relaxing the negatively supercoiled DNA. However, DMSO is already present in the PCR buffer but still, we can use extra DMSO if amplification is not obtained (mainly in high GC rich DNA).
Moreover, it is even useful in cell proliferation, inhibition and differentiation studies and as a crypto preservative as well.
Apart from these advantages, DMSO has one major disadvantage. Boosting PCR amplification is not always useful, as it increases the mispairing of bases. This causes mutagenesis.
An inappropriate amount of DMSO facilitates the flexibility to primer templet binding. Also, it boosts activity of Taq DNA polymerase. henceforth, increasing the amount of DMSO induces mutagenesis during amplification.
Conclusively, it is not a good practise if you are sending the amplicons for DNA sequencing. So what is the exact concentration of DMSO for PCR reaction?
My ultimate guide for using DMSO in PCR:
You have to optimize your own reaction. Don’t worry I will tell you how.
If the GC content is more than 60%, it is considered as a highly sensitive reaction. You have to try three or four different combinations of DMSO for that. Ideally, use 5.5%, 6% or 7% of DMSO in three different reactions. Analyse the result and decide which combination is good for your reaction.
Addition of DMSO decreases the annealing temperature of the PCR reaction. So if you have two reactions at once. For example, one with annealing temperate of 60°C and other with 63°C by adding DMSO to the reaction of 63°C temperature we can reduce the annealing temperature of that reaction. Ultimately, we can run both reactions at the same annealing temperature of 60°C.
However, for doing this type of stuff, you need expertise and in-depth knowledge of ingredients. You have to run many different PCR experiments to master your PCR skills.
Remember, amplification is the first step of any experiment. you’ve to learn how to play with components, otherwise, every time you have to relies on ready to use buffers.
Our motto of writing these type of content is to give you some expert touch and boost your PCR skills.
DMSO is good for PCR. It decreases the melting temperature, increases the specificity and prevents the secondary structure formation. Although, for DNA sequencing, it is not recommended due to int’s mutagenesis activity.
If you are amplifying DNA for sequencing, please prefer to use ready to use PCR enhancer buffer.