PCR Polymerase Chain Reaction Steps and Applications

PCR Polymerase Chain Reaction steps and applications post briefly explains about Polymerase Chain Reaction (PCR) principle, Requirements for PCR, Steps Involved in PCR, and Advantages.

1. Principle of PCR

2. Requirements for PCR

3. Steps Involved in PCR

4. Advantages of PCR

Principle of PCR

Polymerase chain reaction, or PCR, is a method used in the lab to quickly make millions or billions of copies of a specific section of DNA so that it can be studied in more detail. In PCR, short pieces of synthetic DNA called “primers” are used to choose a part of the genome to be amplified. This part is then amplified through multiple rounds of DNA synthesis. There are several applications for the polymerase chain reaction (PCR) in a wide range of fields in both biology and medicine.

Requirements for PCR

Taq Polymerase

PCR uses a DNA polymerase enzyme that replicates new DNA strands from existing DNA strands, just like an organism’s DNA replication. Taq polymerase, named after the heat-tolerant bacteria (Thermus aquaticus) from which it was originally isolated, is the most often used DNA polymerase in PCR.

The optimal temperature for the activity of this heat-stable DNA polymerase is approximately 70 degrees Celsius (high enough for DNA polymerase in humans or E. coli to stop working). Due to its resistance to heat, Taq polymerase is an excellent choice for PCR.

PCR primers

Primer, a short nucleotide sequence used to initiate DNA synthesis. For example, the researcher can choose which regions of DNA will be replicated or amplified by the primers that she or he has selected.

Primers for polymerase chain reaction (PCR) are short pieces of single-stranded DNA, usually about 20 nucleotides long. Each PCR reaction employs two primers, which are designed to surround the region of interest.

In other words, they are given sequences that cause them to attach to opposite strands of the template DNA at the boundaries of the region to be copied. The primers bind to the template via complimentary base pairing.

When the primers are attached to the template, the polymerase can make them longer, copying the area between them.

Steps Involved in PCR

Taq polymerase, primers, template DNA, and nucleotides are the four essential components that make up a PCR reaction (DNA building blocks). In order to manufacture DNA, the necessary components are mixed together in a tube, along with any cofactors that the enzyme may require. The contents of the tube are then subjected to a series of heating and cooling cycles.

These are the fundamental steps in PCR:

Denaturation: Strong heating (96˚C) of the process is needed to denature the DNA strands, which will allow them to be separated. This then produces a template consisting of a single strand for the subsequent step.

Annealing: Annealing reduce the temperature (55˚C – 65˚C) of the process so that the primers can bind to the single-stranded DNA Template at their appropriate complementary sequences.

Extension: During the extension step, the reaction temperatures (72˚C) are increased so that Taq polymerase may extend the primers and produce new strands of DNA.

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In a normal PCR reaction, this cycle is repeated anywhere from 25 to 35 times. The total time required for the reaction might range anywhere from 2 to 4 hours, depending on the length of the DNA region that is being copied. It is possible for the target region to go from having just one or a few copies to having billions of copies if the reaction is efficient.

This is because the original DNA is not used as a template every time. Instead, the newly synthesised DNA can be used as a template for the next round of DNA synthesis. The amount of DNA molecules can roughly double in each round of cycling since there are numerous copies of the primers and many molecules of Taq polymerase floating about in the process. The graphic below illustrates this exponential growth pattern.

PCR Polymerase Chain Reaction steps and applications

Polymerase Chain Reaction (PCR) Steps

Visualization

Gel electrophoresis is the technique that is typically utilised in order to view, or make visible, the results of a PCR reaction. Gel electrophoresis is a method for separating DNA fragments according to size. In this method, DNA fragments are separated by being dragged thru a gel matrix by an electric charge.

PCR Polymerase Chain Reaction steps and applications

A DNA ladder or DNA marker is commonly used to determine the size of the PCR fragments. If the gel is marked with a DNA-binding dye such as ethidium bromide (EtBr), the DNA fragments of the same length will form what is known as a “band,” which is visible to the naked eye on the gel.

Advantages of PCR

A DNA sequence can be amplified by PCR an enormous number of times millions or even billions of times which results in the production of sufficient DNA copies for further analysis. For example, the DNA may be read out using gel electrophoresis, sent out to be sequenced, or processed with restriction enzymes before being cloned into a plasmid. All of these methods are possible.

Numerous research facilities make use of polymerase chain reaction (PCR), which also has practical uses in the fields of forensics, genetic testing, and diagnostics.

It is also possible to use PCR to screen for the presence of bacteria or DNA viruses in a patient’s body: if the pathogen is present, it may be possible to amplify portions of the pathogen’s DNA from a blood or tissue specimen.

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