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PCR is a technique used in the lab to make millions of copies of a item section of DNA. It was first developed in the 1980s.

What is PCR?

The polymerase chain reaction (PCR) 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 brand many copies of (dilate) pocket-sized sections of DNA or a gene.
Using PCR it is possible to generate thousands to millions of copies of a particular section of DNA from a very modest amount of DNA.
PCR is a common tool used in medical and biological inquiry labs. It is used in the early stages of processing DNA for sequencing, for detecting the presence or absenteeism of a cistron to help identify pathogens during infection, and when generating forensic Dna profiles from tiny samples of Deoxyribonucleic acid.

How does PCR work?

The principles backside every PCR, whatever the sample of Deoxyribonucleic acid, are the same.
Five core 'ingredients' are required to fix up a PCR. Nosotros will explain exactly what each of these do as we keep. These are:
- the DNA template to exist copied
- primers, short stretches of DNA that initiate the PCR reaction, designed to demark to either side of the section of Deoxyribonucleic acid y'all want to copy
- Dna nucleotide bases (also known as dNTPs). Dna bases (A, C, Chiliad and T) are the building blocks of Dna and are needed to construct the new strand of Dna
- Taq polymerase enzyme to add in the new Dna bases
- buffer to ensure the right weather for the reaction.
PCR involves a process of heating and cooling called thermal cycling which is carried out by motorcar.
There are three main stages:
1. Denaturing – when the double-stranded template DNA is heated to split it into ii unmarried strands.
2. Annealing – when the temperature is lowered to enable the Deoxyribonucleic acid primers to attach to the template DNA.
3. Extending – when the temperature is raised and the new strand of Deoxyribonucleic acid is made by the Taq polymerase enzyme.
These 3 stages are repeated 20-40 times, doubling the number of Deoxyribonucleic acid copies each time.
A complete PCR reaction tin can be performed in a few hours, or even less than an hr with certain high-speed machines.
Later PCR has been completed, a method called electrophoresis tin can be used to check the quantity and size of the DNA fragments produced.

Illustration showing the main steps in the polymerase chain reaction (PCR).

Illustration showing the main steps in the polymerase chain reaction (PCR). Paradigm credit: Genome Research Express

What happens at each stage of PCR?

Denaturing stage

During this stage the cocktail containing the template Deoxyribonucleic acid and all the other core ingredients is heated to 94-95⁰C.
The high temperature causes the hydrogen bonds between the bases in 2 strands of template DNA to break and the 2 strands to separate.
This results in ii single strands of DNA, which will human activity as templates for the production of the new strands of DNA.
It is of import that the temperature is maintained at this stage for long plenty to ensure that the DNA strands accept separated completely.
This commonly takes between 15-30 seconds.

Annealing stage

During this stage the reaction is cooled to l-65⁰C. This enables the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding (the exact temperature depends on the melting temperature of the primers you are using).
Primers are single strands of Deoxyribonucleic acid or RNA sequence that are effectually 20 to 30 bases in length.
The primers are designed to be complementary in sequence to short sections of DNA on each end of the sequence to exist copied.
Primers serve equally the starting point for DNA synthesis. The polymerase enzyme can only add together Deoxyribonucleic acid bases to a double strand of DNA. Only once the primer has spring tin can the polymerase enzyme adhere and beginning making the new complementary strand of DNA from the loose DNA bases.
The two separated strands of Dna are complementary and run in opposite directions (from 1 end - the 5' stop – to the other - the 3' end); as a issue, there are two primers – a forward primer and a reverse primer.
This stride usually takes well-nigh 10-thirty seconds.

Extending stage

During this final pace, the heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.
Taq DNA polymerase is an enzyme taken from the heat-loving bacteria Thermus aquaticus.
- This bacteria commonly lives in hot springs and so can tolerate temperatures higher up 80⁰C.
- The leaner's Deoxyribonucleic acid polymerase is very stable at high temperatures, which means it tin withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
- Deoxyribonucleic acid polymerase from most other organisms would not be able to withstand these high temperatures, for example, human polymerase works ideally at 37˚C (body temperature).
72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. Information technology attaches to the primer and then adds Deoxyribonucleic acid bases to the single strand one-by-ane in the 5' to 3' direction.
The issue is a make new strand of Dna and a double-stranded molecule of Deoxyribonucleic acid.
The duration of this step depends on the length of DNA sequence beingness amplified but usually takes around one minute to re-create 1,000 DNA bases (1Kb).

These iii processes of thermal cycling are repeated 20-forty times to produce lots of copies of the DNA sequence of interest.
The new fragments of DNA that are made during PCR besides serve every bit templates to which the DNA polymerase enzyme can adhere and start making DNA.
The result is a huge number of copies of the specific Dna segment produced in a relatively short menstruation of time.

Illustration showing how the polymerase chain reaction (PCR) produces lots of copies of DNA.

Illustration showing how the polymerase chain reaction (PCR) produces lots of copies of Deoxyribonucleic acid. Paradigm credit: Genome Research Limited

This page was last updated on 2022-07-21