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 originally 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 very small amount of DNA.
PCR is a common 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 exactly what each of them does as we move forward.
- The DNA template that is going to be copied.
- Primers, 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 basic components of DNA and are needed to build the new DNA chain.
- Enzyme Taq polymerase to be added in the new DNA buffer bases to ensure adequate conditions for the reaction.
- PCR involves a heating and cooling process called thermal cycling that is carried out by machine.
There are three main stages
When the double-stranded template DNA is heated to separate it into two simple chains.
When the temperature is lowered to allow the DNA primers to bind to the template DNA.
When the temperature increases and the new strand of DNA is produced by the enzyme Taq polymerase. These three stages are repeated 20 to 40 times, doubling the number of DNA copies each time.
A complete PCR reaction can be performed in a few hours, or even less than an hour, with certain high-speed machines.
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?
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 the production of the new DNA strands. It is important that the temperature is maintained at this stage long enough to ensure that the strands of DNA have completely separated.
This usually takes between 15-30 seconds.
During this step, the reaction is cooled to 50-65 ° C. 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 melting temperature of the primers you are using).
The primers are simple strands of DNA or RNA, which are about 20 to 30 bases in length. The former are designed to be complementary in sequence to short sections of DNA at each end of the sequence 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. Only 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.
During this final step, the heat increases to 72 ° C to allow the new DNA to be manufactured by a special Taq DNA polymerase enzyme that adds DNA bases.
Taq DNA polymerase is an enzyme taken from heat-loving bacteria. This bacterium normally lives in thermal waters, so it can 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 then adds DNA bases to the individual strand one by one 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 that are produced during PCR also serve as templates to which the enzyme DNA polymerase can bind and begin to produce DNA.
The result is a large number of copies of the specific DNA segment produced in a relatively short period of time.