In the 1970s, the DNA testing process was unsurprisingly not as advanced as it is today. Instead, the PCR reaction had to be reset 4-5 times a day to achieve the desired result. This process was too tedious and had to be refined.
But then came the Taq DNA polymerase, which was isolated from the Thermus Aquaticus, a bacterium found in high-temperature conditions. This isolation didn’t mean much. Not until Kary Mullis used it to develop the PCR method in 1983.
MBP Inc provides high-quality molecular biology products, and we have written this blog about the polymerase chain reaction and explained how it works.
A Look Into The Polymerase Chain Reaction (PCR) [1]
What is PCR?
The polymerase chain reaction was developed and refined by Kary Mullis in 1983 and put out for commercial use in 1988. The primary purpose of the PCR method is to take small DNA samples and create a reaction that makes them multiply. Through the PCR method, you can take a DNA sample and create millions of copies. Even a tiny piece can be worked with. That is what’s so great about the PCR method.
PCR is mainly used for gene sequencing and detecting the presence or absence of specific genes in a sample of DNA.
How Does It Work?
5 Components Required:
Five things are required to ensure a successful PCR reaction. These 5 elements are as follows:
- The DNA template needs to be replicated.
- Primers that will bind to either side of the DNA strands of the template.
- dNTPs (Also known as DNA nucleotide bases). These are the foundational elements of DNA and are essential to the replication process.
- Taq Polymerase Enzyme to optimize the reaction.
- The use of a buffer to recreate the environmental conditions needed for the reaction.
The Thermal Cycling Process:
The first step is the thermal cycling process. In this process, the rapid heating and cooling of the sample are carried out in a three-part process.
Denaturing – In this part, the double-stranded DNA is isolated into two individual strands of DNA. This is done by taking the template DNA and heated to temperatures near 95 degrees celsius or just above 200 degrees Fahrenheit. This causes the bond between the double-stranded DNA to break. This process takes a maximum of 30 seconds, during which the temperature should be maintained.
Annealing – Then come in the primers, which are attached to the singular DNA strands as they cool down. This is done by cooling the reaction down to 50-65⁰C. The primers come in and attach through hydrogen bonding, which is allowed at this temperature.
Primers are the guidance point. They show where the DNA synthesis process should begin from. Primers are essential because, without them, the DNA can’t be replicated.
These primers attach to either side of the DNA strand in opposite directions. They can be differentiated because one is a forward primer, and the other is a reverse primer. This step takes around 10-30 seconds, depending on the primers you are trying to attach.
Extending – In the final step, the heat is increased again—this time to an exact temperature of 72 Degrees Celsius. And the Taq Polymerase is introduced. That is because it adds the DNA bases essential to the replication process.
The Taq polymerase is helpful because it has the Thermus Aquaticus bacterium, which can withstand high temperatures. This process would not have been possible with any other bacterium as they can’t bear such high temperatures.
This entire thermal cycling process is repeated multiple times. Depending upon the sample size required, it can be repeated up to 40 times, creating millions of copies of the DNA sequence. Overall, this process takes around an hour to do carefully.
Conclusion
The PCR method has refined and made gene sequencing very simple. It has also allowed for quick testing of Covid-19, which has made dealing with the virus much easier.
MBP Inc has PCR Plates and PCR reagents in stock for those looking to purchase. Check out the products we offer and order today!
