PCR Full Form

Full form for PCR is polymerase chain reaction. It  is a simple and low-cost method for “amplifying” – or copying – tiny portions of DNA. Because molecular and genetic investigations require considerable volumes of DNA, studies of isolated fragments of DNA are almost impossible without PCR amplification.

PCR, which transformed the study of DNA to such an extent that its originator, Kary B. Mullis, was awarded the Nobel Prize in Chemistry in 1993, is often hailed as one of the most important scientific achievements in molecular biology.

The polymerase chain reaction (PCR) is a typical laboratory technique for making multiple copies of a specific area of DNA (millions or billions!). This DNA region can be whatever the researcher wants it to be. It may be a gene that a researcher wants to learn more about, or a genetic marker that forensic scientists use to link crime scene DNA with suspects.

The purpose of PCR is usually to create enough of the target DNA area to be examined or used in another way. For example, PCR-amplified DNA can be sequenced, visualised through gel electrophoresis, or cloned into a plasmid for further research.

PCR is used in a variety of fields in biology and medicine, including molecular biology, medical diagnostics, and even some aspects of ecology.

PCR, like DNA replication in an organism, necessitates the use of a DNA polymerase enzyme to create new DNA strands from existing ones. Taq polymerase, the DNA polymerase most commonly employed in PCR, is named for the heat-tolerant bacteria from which it was obtained (Thermus aquaticus). Aquaticus is a specie that can be found in hot springs and hydrothermal vents. Its DNA polymerase is extremely heat-stable at a temperature on which a human or E. coli DNA polymerase would be nonfunctional. Taq polymerase is good for PCR because of its thermal stability. As we’ll see, high temperatures are frequently utilised in PCR to denature or separate the template DNA’s strands.

Uses of PCR

The DNA produced by PCR can be used in a variety of laboratory techniques once it has been amplified. The Human Genome Project (HGP), for example, depended heavily on PCR for most mapping techniques.

DNA fingerprinting, detection of germs or viruses (especially AIDS), and identification of genetic abnormalities are only a few of the laboratory and clinical applications of PCR.

How does PCR work

To use PCR to amplify a segment of DNA, the sample is heated until the DNA denatures, or separates into two single-stranded DNA pieces. Then, using the original strands as templates, an enzyme called “Taq polymerase” synthesises – or creates – two new strands of DNA. This process causes the original DNA to be duplicated, with one old and one new strand of DNA in each of the new molecules. After that, each of these strands can be used to make two more copies, and so on. Denaturing and synthesising new DNA is repeated 30 or 40 times, resulting in almost one billion precise copies of the original DNA strand.

The entire PCR cycle procedure is automated and takes only a few hours to complete. It’s controlled by a thermocycler, which is set to change the temperature of the reaction every few minutes to allow DNA denaturation and synthesis.

Taq polymerase, primers, template DNA, and nucleotides are the main components of a PCR reaction (DNA building blocks). The components, together with cofactors required by the enzyme, are combined in a tube then heated and cooled repeatedly to allow DNA to be produced.

Stages of PCR

The basic steps are as follows:

1. Denaturation: When the reaction is heated to a high temperature, the DNA strands are separated, or denatured. For the next stage, you’ll need a single-stranded template.
2. Annealing: Allow the primers to bind to their corresponding sequences on the single-stranded template DNA by cooling the process.
3. Extension: Raise the reaction temperature such that Taq polymerase stretches the primers and synthesises additional DNA strands.

In a normal PCR reaction, this cycle repeats 25 to 35 times, taking 2 to 4 hours depending on the length of the DNA area being copied. The target region can grow from one or a few copies to billions if the reaction is efficient (functions well).

This is due to the fact that the original DNA isn’t used as a template every time. Instead, the new DNA created in one cycle can be used as a template in the subsequent DNA synthesis round. 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 around in the process. The graphic below depicts this exponential growth pattern.

Limitations of PCR

One of the biggest drawbacks of PCR is that it requires prior knowledge of the target sequence in order to build the primers that would allow selective amplification. This means that PCR users usually need to know the exact sequence(s) upstream of the target region on each of the two single-stranded templates in order for the DNA polymerase to appropriately attach to the primer-template hybrids and create the full target region during DNA synthesis.

DNA polymerases, like all enzymes, are prone to errors, which result in mutations in the PCR fragments created.

Another drawback of PCR is that it might amplify even the tiniest quantity of contaminated DNA, resulting in erroneous or unclear results. Separate rooms should be set up for reagent preparation, PCR, and product analysis to reduce the risk of contamination. Single-use aliquots of reagents should be used. Disposable plunger pipettors with extra-long pipette tips should be used on a regular basis.

Conclusion

The use of genotyping techniques to all living species has made great progress in the reconstruction of life’s history. At the population level, the distribution and frequency of known genetic polymorphisms in a species can expose the impacts of natural selection and infer demographic change by highlighting the changing processes at work. Furthermore, the molecular phylogenies that currently dominate classification are based on the comparison of sequences of the same genes between different species as well as whole genomes. They allow scientists to trace the links between species by looking at how their DNA sequences diverge. As a result, the PCR is an important stage on two levels. The first is concerned with the isolation and characterisation of homologous genes in various animals. The creation of amplified total genomic DNA for genome sequencing and comparative analysis is the second step. PCR, on the other hand, is used to determine the genetic heritage of missing creatures. Following the death of the body, DNA fragmentation occurs. If we can recover and amplify these fragments, we can deduce all or part of the individual’s initial genome, regardless of its current status. In the discipline of paleogenetics, which entails recovering and interpreting DNA sequences from more or less ancient creatures, PCR has thus become the fundamental method.