The role of DMSO in PCR amplification is to increase the specificity and yield of PCR. DMSO is a PCR enhancer which is an important ingredient of PCR buffer. In this article, we will discuss the structure and function of DMSO and how we can utilize DMSO for enhancing the PCR reaction and finally, my ultimate guide for using DMSO.
The PCR buffer is used to enhance a PCR reaction made up of MgCl2, KCl, albumin, (NH4)2 SO4 and DMSO. However, the manufacturer never reveals the exact concentration and component of their buffer.
Each of the listed components is specifically utilized to perform a specific function. For example, the MgC2 work as the cofactor of Taq DNA polymerase. For more detail on MgCl2 and Taq DNA polymerase please read our articles:
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 DMSO in PCR because it is used in some special type of PCR reactions. One of the special types of PCR reaction is high GC rich PCR.
GC content of templet DNA is one of the causes of failure in PCR reaction. The high GC content results in reaction failure, non-specific bindings or non-confirmative results.
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. The high temperature is required to break the triple bonds between G and C and due to higher stability, it leads to secondary structure formation during PCR.
Additionally, it causes a non-specific binding of primer results in the non-confirmative result. To overcome these problems, DMSO is added to the PCR buffer.
Role of DMSO in PCR
DMSO makes GC rich DNA more heat labile and reduces the Tm of reaction. Here, DMSO directly binds to the Cystine residue of the GC rich region and changes the conformation of Cystine 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 decreases the denaturation temperature.
DMSO even prevents the secondary structure formation. Due to the high GC content in DNA, the DNA creates the secondary structure or hairpin loop. Three hydrogen bond require more energy to broke, the secondary structure is formed by binding of single-stranded DNA with each other, just like a hairpin loop.
This will result in PCR failure. DMSO binds to the DNA and prevents the reannealing of single-stranded DNA. It also facilitates the annealing of primer with templet. Therefore, it increases the specificity and yield of PCR reaction.
Generally, the GC content of the 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.
Further, it depends on the type of reaction, the GC content of DNA and the quality of DMSO.
Beside increasing specificity, it changes the DNA topology as well. DMSO releases supercoiling of DNA which is confirmed by plasmid studies. Here, DMSO greatly induces the activity of topoisomerase I. The topoisomerases are the enzyme which helps in relaxing the 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).
Besides use in molecular genetics, it is even applicable 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 will induce mutagenesis.
The DMSO incorporates mutations in the amplicon by mismatch base pairing. An inappropriate amount of DMSO facilitates the flexibility to primer templet binding, the activity of Taq polymerase is greatly increased results in mismatch base-pairing. Hence the rate of mutation is increased by increasing the concentration of DMSO.
So what is the exact concentration of DMSO for PCR reaction?
My ultimate guide for using DMSO in PCR
Based on the available research data, 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, for optimizing this reaction, you have to optimize the PCR by adding three of four types of different combination of DMSO. 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. Interestingly, you can use DMSO artistically. We are a scientist but we are also an artist. We can run more than 2 reactions on the same annealing temperature.
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 and ultimately, we can run both reactions at the same annealing temperature of 60°C.
However, for doing this, you need to have expertized and special skills regarding PCR. You have to perform so many experiments at once for mastering these types of skill. Once you know about each and every component used in PCR, you can do it easily.
Therefore, our motto of writing this types of content is to give you expertize theoretically through our personal research experience. If you have any question regarding the topic or want or know more please comment in the comment box.
Article covered by: Tushar Chauhan
Article reviewed by: Ravi Parmar