History of PCR:
The invention of PCR is typically credited to Kary Mullis, a technician at Cetus Corporation tasked with enhancing oligonucleotide synthesis. In his 1969 Nobel Prize lecture, he discusses how, while camping with his partner, he had the idea for PCR.
For molecular and genetic testing, enormous amounts of DNA are often required, but the PCR technique enables scientists to produce millions of copies from a very tiny amount of DNA. There are numerous uses for the PCR method, which is frequently used in biological and medical research laboratories.
On the other hand, a “PCR test” is often a rapid, precise diagnostic test for the early stages of an infectious disease. For instance, this is one method of detecting COVID-19’s causative virus, SARS-CoV-2.
Define PCR.
The highly sensitive PCR method enables quick DNA amplification of a particular section. Using visual methods based on size and charge, PCR may detect and identify gene sequences by producing billions of copies of a certain DNA fragment or gene.
Thermocyclers, DNA amplifiers, or heat cycles are other names for PCR machines. Small genomic DNA or RNA segments are amplified by PCR equipment using a primer. It is a cheap and highly efficient tool. It operates on the principles of complementary nucleic acid hybridization and nucleic acid replication to produce a given target DNA/RNA sequence exponentially by a factor of 107 in a few hours.
In research labs and clinical diagnostics, PCR machines are used to replicate DNA, detect DNA sequences, perform DNA fingerprinting, forensic analysis, and molecular cloning, diagnose genetic diseases, and detect pathogens like the Hep B and C viruses, HIV-1 that causes AIDS, Chlamydia trachomatis, Mycobacterium tuberculosis, Human Papillomavirus, and Cytomegalovirus. This accelerates other temperature-sensitive procedures like digestion using restriction enzymes or quick diagnostics. These employ the polymerase chain reaction to amplify DNA fragments (PCR).
One of the most important scientific advancements in molecular biology is PCR, also referred to as “molecular photocopying.” As a result of its revolutionary impact on DNA research, Kary B. Mullis, the invention’s creator, received the Chemistry Nobel Prize in 1993. Since significant amounts of sample DNA are needed for molecular and genetic investigations, examining isolated DNA fragments without PCR amplification is almost impossible.
Principle of PCR:
The DNA polymerase enzyme uses a single-stranded DNA template as a scaffold to direct the synthesis of DNA from deoxynucleotide substrates. when a longer template DNA is annealed to an oligonucleotide that has been specially designed. The 3′ end of the DNA molecule receives nucleotides from DNA polymerase. Thus, when annealed to a single-stranded template containing a region corresponding to the oligonucleotide, DNA polymerase can use a synthetic oligonucleotide as a primer and extend its 3′ ends to form a long stretch of double-stranded DNA.
Explain the parts of PCR.
- The thermal block of the thermocycler contains openings where tubes containing the reaction mixtures can be placed.
- A heated lid that is pushed against the reaction tube lids is included. The lid stops water from the reaction mixtures from condensing on the inside of the lids.
- Rotation of the lid knob clockwise lowers the heating plate that pressurizes the cap of the tubes to seat firmly in the block and ensure greater contact. In contrast, turning it in a clockwise direction raises the lid, allowing users to slide it to the back.
- A control panel has a Key-Pad for entering different protocols and settings as well as a sizable graphical display that makes it easy to read and shows the status of the many system features and functions.
- Air output and air intake are made easier by the front, lateral, and bottom air vents.
PCR Components(PCR reagent):
DNA Template:
- Selecting the proper PCR templates is essential.
- In contrast to classical PCR, which uses DNA templates, RT-qPCR uses RNA templates to make complementary DNA.
- As a result, the usage of a trustworthy PCR template preparation kit is necessary for the first step of a successful PCR.
Thermostable DNA polymerase:
- All PCR reactions require a DNA polymerase that can operate at a high temperature (about 70 °C), as the first step of PCR requires separating DNA strands at a high temperature (90 °C).
- Taq polymerase, a well-liked DNA polymerase for PCR derived from the thermophilic bacterium Thermus aquaticus, is a heat-stable enzyme.
Oligonucleotide Primers:
- Primers serve as a beginning point for the DNA/RNA polymerase to start synthesizing new DNA. They are brief strands of nucleotides (DNA or RNA) complementary to the template DNA.
- Primers are brief strands of complementary nucleotides (DNA or RNA) that serve as the DNA/RNA polymerase’s starting point for DNA synthesis. They are complementary to the template DNA. They are required for DNA synthesis to begin. Primer annealing to single-strand DNA requires lower temperatures (50–65°C) than denaturation, which produces hydrogen bonds once annealing is complete
Deoxyribonucleotide triphosphate(dNTPs):
- For DNA polymerase to be able to manufacture DNA, dNTPs are necessary.
Buffer System:
- PCR buffers ensure that the ideal conditions are always kept for the PCR process.
- The PCR buffer mainly consists of Tris-HCl, potassium chloride (KCl), and magnesium chloride (MgCl2).
- Tris-HCl and KCl are used to keep the pH constant during the PCR process. Magnesium ions act as cofactors for DNA polymerase, ensuring that DNA synthesis occurs properly during PCR.
Explain the steps of PCR.
Denaturation:
- Denaturation occurs when the reaction mixture is heated for 0.5 to 2 minutes to 94°C.
- When the hydrogen bonds between the DNA’s two strands are disrupted, single-stranded DNA is produced.
- The single DNA strands are now used as a template to create new DNA strands.
Annealing:
- The reaction temperature is dropped to 54 to 60 °C for around 20 to 40 seconds.
- In this case, the primers bind to complementary sequences in the template DNA.
- Primer sequences are single-stranded DNA or RNA segments that are 20–30 nucleotides long.
- They serve as the starting point for the creation of DNA.
- The two separated strands are primed using a forward primer and a reverse primer, which run in opposite directions.
Elongation:
- At this point, the temperature is raised to a range of 72 and 80 degrees Celsius.
- The nucleotides are attached to the 3′ ends of the primer by the Taq polymerase enzyme. The outcome is that the DNA extends from 5′ to 3′.
- Taq Polymerase can survive temperatures that are very high. As a result, a DNA molecule with two double strands is created.
These three steps are repeated 20–40 times in order to quickly extract multiple DNA sequences of interest.
What is the working process of PCR?
- The sample is first heated in a PCR machine, which separates the DNA into two pieces of single-stranded DNA and causes the denaturation of the sample.
- Then, using the existing DNA strands as templates, an enzyme called “Taq polymerase” builds two new DNA strands.
- This process duplicates the original DNA, resulting in each new molecule having both the original and duplicated strands of DNA.
- Following that, two further copies can be made using each of these strands, and so on.
- When the cycle of denaturing and synthesis of new DNA is repeated up to 30 or 40 times, there are more than one billion identical copies of the original DNA segment.
- The entire automated PCR cycle process can be completed in a matter of hours. The reaction is managed using a tool called a thermocycler, which is set up to alter the reaction’s temperature every few minutes in order to promote DNA synthesis and denaturation.
What are the applications of PCR?
Gene transcription:
- Using PCR, it is possible to look into how different cell types, tissues, and species express their genes differently throughout time.
- cDNA is produced using reverse transcription once RNA from relevant samples has been isolated.
- The amount of cDNA generated by PCR can then be used to determine the original RNA concentrations for a certain gene.
Genotyping:
- It is possible to identify sequence variations in particular cells’ or organisms’ alleles.
- The amplification of a mutation or a transgenic component is made easier by genotyping transgenic organisms.
Cloning and mutagenesis:
- By inserting amplified dsDNA pieces into vectors including gDNA, cDNA, and plasmid DNA, PCR cloning enables the breeding of novel bacterial strains with altered genetic makeup.
- Cloning helps introduce point mutations by site-directed mutagenesis, which uses the recombinant PCR technique on its own.
- It also helps to produce unique gene fusions.
Sequencing:
- After template DNA has been amplified, purified, and processed through a sequencing phase, sequencing takes place.
- During the library preparation stage of next-generation sequencing (NGS), PCR is also used to quantify DNA samples and tag them with sequencing adaptors for multiplexing.
Medicine and biomedical research:
- Medical applications range from recognizing infectious organisms to genetic changes related to the disease. Prenatal genetic testing uses a polymerase chain reaction to identify chromosomal abnormalities and genetic mutations during pregnancy, giving expectant parents vital information about the propensity of their offspring to develop a particular genetic disease.
- It can also be used to screen embryos for in vitro fertilization using a preimplantation genetic diagnostic procedure (IVF).
Forensic Science:
- PCR can be helpful in paternity tests and forensic investigations to determine the sources of samples.
- It is used to amplify DNA from artifacts in molecular archaeology.
Environmental microbiology and food safety:
PCR can be used to identify pathogens in matrices like food and water in addition to patient samples. This is crucial for both identifying infectious diseases and preventing them.
What are the advantages and disadvantages of PCR?
Advantages:
- Enables more rapid and well-informed decision-making
- rapid detection of bacteremia, particularly in cases where the bacterial count is low
- Effective in finding extrapulmonary instances that smear and/or culture might have missed.
- Important for identifying specific diseases that are difficult or take a long time to culture in vitro. results significantly more quickly than cultivating
- For some diagnostic procedures that rely on smear and culture, including tuberculosis, it is still regarded as an adjunct test.
- It can perform an antimicrobial resistance test.
Disadvantages:
- PCR does not allow for the amplification of unknown targets. Prior knowledge of the target sequence is necessary for the primer designs.
- Mutations in the PCR product are a potential consequence of error-prone DNA polymerases.
- PCR is particularly prone to contamination. If there is even a small bit of tainted DNA, the results could be misconstrued or confused.
- PCR efficiency falls as amplicon size increases.
References:
https://microbenotes.com/pcr-machine-principle-parts-steps-types-uses-examples/
