PCR (Polymerase Chain Reaction)

PCR (Polymerase Chain Reaction)

The polymerase procedure or PCR testing is a scientific lab methodology for replicating DNA that facilitates the precise amplification of a “targeted” DNA pattern. In only a couple of hours, the PCR test procedure can take the tiniest quantity of DNA material and then clone multiple times and replicate it to millions. 

An American biochemist, Kerry Banks Mullis, first devised the procedure of PCR in 1985, and it has since proven to be a vital and common instrument in numerous biological lab investigations.

What do we understand by PCR?

In genetic biochemistry, PCR can create numerous clones of tiny portions of DNA or genes.

PCR may create hundreds to millions of replicas of a specific sequence of DNA using a relatively tiny quantity of DNA.

In biomedical and pharmacological scientific labs, polymerase chain reaction (PCR) is a favoured technique. It is employed through the initial stages of Genome synthesis for sequence analysis, identifying the existence or lack of a genetic to aid identification of microorganisms following infections and creating forensic DNA patterns using trace amounts of DNA.

The principles on which a PCR takes action

A PCR process is centered on the scientific process of DNA replication by enzymes. PCR uses primer catalysts to amplify the growth rate of a small piece of DNA.

Polymerases create synthetic DNA sequences that are equivalent to the parent DNA strand. Just the already present 3′-OH ring gets a new nucleotide which may get added onto the DNA polymerase. 

As a result, a primer becomes necessary. And additional nucleotides get incorporated into the DNA polymerase’s primary end.

5 Core Components Required to Set up a PCR 

A PCR test requires five main ‘components’. As we proceed forward, we’ll illustrate how each one of these components performs. They are as follows:

  1. The initial or primary DNA sequence for replication
  2. Primers are small lengths of DNA that start the process. These get intended to attach on either side of the piece of DNA, which then gets replicated.
  3. DNA nucleotide foundations- The prime structural components of DNA are required to make the replicated DNA strands.
  4. Taq polymerase catalytic enzymes get used to incorporate the additional DNA nucleotides.
  5. A buffering component to guarantee that the reaction takes place under the proper circumstances

Thermal cycling is the procedure of continuously rising and lowering the temperature during the entire process. This procedure gets carried out by a piece of machinery in a PCR.

Once these components are ready and put together, the PCR process commences into its different stages. 

The different stages of a polymerase chain reaction

Three main cyclic events are involved in the entire PCR:

Denaturation

  • As the reaction combination gets heated to about the temperature of 94°C for around 0.5 to 2 mins, the stages of denaturation start. The H2 bonds connecting two DNA strands are broken, resulting in two single-stranded DNA sequences.
  • The individual segments of DNA are now used as a blueprint for the creation of more DNA segments. To guarantee the splitting of the double-stranded sequence, the degree of temperature must be stably maintained for a prolonged period.

Annealing

  • Now over about 20-40 secs, overall process temperature gets reduced to 54-60°C. The markers attach to each corresponding section on the target DNA during this stage.
  • These primers are all single-stranded DNA and RNA strings of around 20-30 nucleotides in size.
  • These act as the beginning site in the DNA replication process.
  • Because the separate split segments flow in opposing directions, there are two specific primers: a forwards primer and a reversal primer.

Elongation

  • At this stage, it is again heated, and the temperature gets increased up to 72-80°C during this point. The Taq polymerases add the nucleotides towards the 3′ terminus of the DNA template.
  • It causes the DNA strand to lengthen inside this 5′ to 3′ direction. Given optimal circumstances, the Polymerases add around 1000bp every min.
  • The thermostable DNA polymerase 1 can withstand extreme temperatures. It binds itself to the template and introduces nucleotides to the single DNA segments. And hence, double-stranded DNA sequences are formed.

These three processes mentioned above are performed repeatedly about 20-40 times to get a large quantity of DNA sequencing of relevance within a very brief period.

The different types of PCR Processes

There are several forms of PCR.

PCR in real-time

The DNA replication is observed within true time using a luminous sensor in such a case. The luminous sensor’s output intensity is related to the quantity of the replicated DNA fragments.

Nested PCR

It was conducted to increase sensibility and precision. They decrease non-specific reagent adherence by multiplication of unanticipated prime matching spots.

PCR multiplexing

This method is designed to amplify numerous substrates in a singular PCR process. It concurrently multiplies a variety of DNA sequences.

Quantitative PCR

It detects, characterizes, and quantifies a recognized genome in a specimen using DNA replication uniformity.

PCR using arbitrary primers

This is a PCR-based DNA profiling method. It employs primers whose DNA pattern is selected at random.

There are several other types of PCR procedures, including the following-

  • Long-range PCR
  • Single-cell PCR
  • Fast-cycling PCR
  • Methylation-specific PCR (MSP)
  • Hot start PCR
  • High-fidelity PCR
  • In situ PCR
  • Variable Number of Tandem Repeats (VNTR) PCR
  • Asymmetric PCR
  • Repetitive sequence-based PCR

How are PCR tests applied?

PCR seems to have the potential applications:

Healthcare and medicine

  • Genetic illness mutagenesis testing
  • Monitoring the gene during gene or genotype therapy
  • Finding illness or disorder sourcing genes in parents.
  • Essential for organ transplants

Crime Scene Investigation

  • It is employed in DNA fingerprinting as a tool.
  • Finding the culprit among multitudes of individuals.
  • Paternity examinations

Benefits for contagious infections and diseases

  • Examining clinical diagnostic samples for the existence of infectious pathogenic pathogens such as HIV, hepatitis, malaria, tuberculosis, and so on.
  • The discovery of novel aggressive types and subtypes of organisms that cause outbreaks.

Genetics and research

  • In genetic research, two species’ genomes are compared.
  • Genealogical study of DNA through any origin, including fossils.
  • Genomic profiling
  • Sequencing of Genes

Conclusion

The pragmatic progress of the straightforward yet flexible PCR test has significantly impacted scientific work. The only thing we need to do is combine the PCR ingredients in the proper quantities in a tiny container, feed it inside the PCR equipment, and allow time to do the work. The specialists will then have millions or even billions of replicas of the desired DNA. 

Isn’t it incredible?

X