Index
PCR is a technique used in the laboratory to make millions of copies of a particular section of DNA. It was first developed in the 1980s.
What is PCR?
The polymerase chain reaction, or PCR (for its acronym in English), was initially developed in 1983 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work.
PCR is used in molecular biology to make many copies of (amplify) small sections of DNA or a gene.
Using PCR, it is possible to generate thousands or millions of copies of a particular section of DNA from a minimal amount of DNA.
PCR is a standard tool used in medical and biological research laboratories. It is used in the early stages of DNA processing for sequencing to detect the presence or absence of a gene that helps identify pathogens during infection and to generate forensic DNA profiles from small DNA samples.
How does the polymerase chain reaction work?
The principles behind each polymerase chain reaction, whatever the DNA sample, are the same. Five central “ingredients” are required to establish a PCR. We will explain precisely what each of them does as we move forward.
These are:
- The DNA template is going to be copied.
- Primers are short stretches of DNA that initiate the PCR reaction, designed to join either side of the section of DNA you want to copy.
- Nucleotide bases of DNA (also known as dNTPs). The DNA bases (A, C, G, and T) are the essential components of DNA and are needed to build the new DNA chain.
- Enzyme Taq polymerase is added to the new DNA buffer bases to ensure adequate conditions for the reaction.
- PCR involves a heating and cooling process called thermal cycling carried out by a machine.
There are three main stages.
Denaturation:
When the double-stranded template DNA is heated, separate it into two simple chains.
Annealing:
When the temperature is lowered, the DNA primers can bind to the template DNA.
Extension:
When the enzyme Taq polymerase produces, the temperature increases, and the new strand of DNA, these three stages are repeated 20 to 40 times, doubling the number of DNA copies each time.
A complete PCR reaction can be performed with specific high-speed machines in a few hours or even less than an hour.
Once the PCR is complete, a method called electrophoresis can be used to verify the amount and size of the DNA fragments produced.
What happens at each stage of the PCR?
Denaturing stage:
During this stage, the cocktail containing the template DNA and all the other core ingredients is heated to 94-95⁰C. The high temperature causes the hydrogen bonds between the bases in two chains of DNA to be broken and the two chains to separate.
This results in two simple strands of DNA, which will act as templates for producing the new DNA strands. The temperature must be maintained at this stage long enough to ensure that the strands of DNA have entirely separated.
This usually takes between 15-30 seconds.
Annealing stage:
The reaction is cooled to 50-65 ° C during this step. This allows the primers to adhere to a specific location in the single-stranded template DNA via hydrogen bonds (the exact temperature depends on the primers’ melting temperature).
The primers are simple strands of DNA or RNA, about 20 to 30 bases in length. The former is designed to be complementary in sequence to short sections of DNA at each end of the line to be copied.
The primers serve as a starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Once the primer has been bound, the enzyme polymerase binds and begins to form the new complementary strands of DNA from the loose DNA bases.
The two separate strands of DNA are complementary and run in opposite directions (from one end – the 5 ‘end to the other – the 3’ end); as a result, there are two primers: a forward primer and a reverse primer.
This step usually takes around 10-30 seconds.
Extension stage:
During this final step, the heat increases to 72 ° C to allow the new DNA to be manufactured by a particular Taq DNA polymerase enzyme that adds DNA bases.
Taq DNA polymerase is an enzyme taken from heat-loving bacteria. This bacterium lives typically in thermal waters to tolerate temperatures above 80 ° C.
The DNA polymerase of the bacteria is very stable at high temperatures, which means that it can withstand the temperatures necessary to separate the strands of DNA in the denaturation stage of the PCR.
The DNA polymerase of most other organisms could not withstand these high temperatures; for example, the human polymerase works ideally at 37 ° C (body temperature). 72⁰C is the optimal temperature for the Taq polymerase to build the complementary chain.
It binds to the primer and adds DNA bases to the individual strand in the 5 ‘to 3’ direction.
The result is a new DNA strand and a double-stranded DNA molecule. The duration of this step depends on the length of the DNA sequence being amplified, but it usually takes about one minute to copy 1,000 bases of DNA (1Kb).
These three thermal cycle processes are repeated 20-40 times to produce many copies of the DNA sequence of interest. The new DNA fragments produced during PCR also serve as templates for the enzyme DNA polymerase to bind and begin to make DNA.
Many copies of the specific DNA segment are produced in a relatively short period.
