PCR buffer is a vital component of Polymerase Chain Reaction also called molecular photocopying. The reaction that has helped scientists around the world to study DNA by providing its ample amount from minimal samples. It is almost impossible to carry on a PCR reaction without a buffer. But what is the main function of a PCR Buffer? How does it help in creating DNA copies?
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A PCR buffer is a mixture of compounds that creates a suitable environment for the polymerase chain reaction to proceed. You know that DNA making is a life process, and it needs an environment similar to the cell’s internal conditions to proceed. But if have a PCR master mix then you can carry on this process easily.
During a PCR reaction, technicians face problems if they fail to create the specific conditions needed for the process. Some of these hurdles are unnecessary amplification, non-specific binding, and even reaction failure.
Unexpectedly long primers, improper chemical concentrations, distorted template, or high guanine and cytosine content in template strand, anything can cause reaction failure. PCR reaction buffer helps in overcoming these problems.
Table of Contents
What is PCR?
The polymerase chain reaction is an extensively used laboratory technique. That creates copies of the DNA segment of interest.
It is a widely used application in research, medicine, and forensics. It helps in the diagnosis of genetic disorders by identifying the genes associated with the disease.
It is also a salient tool for forensic investigations. It can help in identifying criminals and establishing paternity.
But to do all of the above procedures, we need an ample amount of DNA.
However, DNA is only a minimal part of the cell. A single cell contains six picograms of DNA. Hence, it will be difficult to extract it in amounts that can be analyzed and experimented with.
So, PCR helps researchers to create exact copies of the DNA segment needed to be tested or analyzed. The freshly prepared sample of deoxyribonucleic acid is identical in composition to the sample extracted. Thus, this process is also known as molecular photocopying.
What is a buffer?
I think we have created enough hype to spark an element of curiosity in you about PCR buffer.
Buffer is an aqueous solution made up of an acid and its conjugate base, holding the capacity to resist any type of pH change of the reaction solution. Thus, it brings stability to reactions that require any specific pH range exactly like molecular photocopying.
One buffer solution cannot facilitate all reactions every reaction has a specific buffer. And every stabilizing solution has its buffer capacity. It is the amount of acid or base that can be added to the reaction mixture before the pH begins to change.
Composition of PCR Buffer:
The composition of a buffer can make your reaction smooth or can turn it into havoc. Hence, it is essential to keep a check on the composition of your PCR buffer.
A PCR buffer is made of many chemical components. Here, we have discussed the main components that are necessary to make a successful buffer.
It is one of the most crucial parts of a buffer that acts as both cofactor and catalyzer. It increases the productivity of Taq Polymerases.
The amount of MgCl2 depends upon the desired results, but it can vary from 0.05 mM to 5.0 mM. It has multiple-functionality in the polymerase chain reaction.
- Increases amplification.
- Prevents denaturation of template DNA by increasing the melting point of DNA.
- By directly binding to the phosphate backbone of the DNA, it prevents denaturation and increases the time for the primer to bind on target.
KCl helps in neutralizing the PCR. It also acts by binding to the DNA backbone like MgCl2. Its composition can vary from 25mM to 100mM. The various functions it performs in molecular photocopying is as follows:
- It is responsible for the elongation of the primer-DNA complex.
- It works best with shorter DNA fragments but can fail when it comes to longer DNA fragments.
Dimethyl Sulfoxide can be a principal part of your PCR buffer if the template DNA segment is abundant in GC. It is difficult to amplify a DNA template having high GC content. Due to its higher chances of formation of the secondary hairpin structure.
- It prevents hairpin structure formation.
- It increases the sensitivity of the primer for binding.
It works as an assistant for the DMSO. It also helps in preventing the formation of secondary structure formation.
This compound is not as popular as any of the chemicals mentioned above. But it perfectly substitutes the DMSO.
Bovine Serum Albumin:
Some DNA samples contain PCR inhibitors such as marine soil, water, or fecal matter. These inhibitors prevent the elongation of the DNA strands.
BSA interferes with the activity of these inhibitors.
PCR buffer is a chemical solution that maintains the ideal environment for a polymerase chain reaction. It stabilizes the pH of the PCR solution.
A buffer is a mixture of an acid and its conjugate base or vise versa. The composition of the buffer depends on the reaction to be carried.