PCR Machine- Principle, Parts, Steps, Types, Uses, Examples

PCR machine is also known as thermal cyclers or DNA amplifiers, or thermocyclers. PCR machines amplify small segments of DNA or RNA chosen from the genome with a primer. It is inexpensive and a very effective tool.

It works under the concepts of complementary nucleic acid hybridization and nucleic acid replication to exponentially manufacture specific target DNA/RNA sequences by a factor of 10^7 in a couple of hours.

PCR machines are used in research labs and clinical diagnostics to replicate DNA, detect DNA sequences, perform DNA fingerprinting, forensic analysis, and molecular cloning, diagnose genetic diseases, and detect pathogens like Hep B and C viruses, HIV-1 causing AIDS, Chlamydia trachomatis, Mycobacterium tuberculosis, Human Papillomavirus, and Cytomegalovirus. This speed up other temperature-sensitive processes like restriction enzyme digestion or rapid diagnostics. These amplify segments of DNA using the polymerase chain reaction (PCR).

PCR, sometimes known as “molecular photocopying’, is frequently hailed as one of the most significant scientific developments in molecular biology. It revolutionized the study of DNA to the point where its inventor, Kary B. Mullis was awarded the Chemistry Nobel Prize in 1993. Without PCR amplification, examinations of isolated fragments of DNA are almost difficult since considerable volumes of sample DNA are required for molecular and genetic analyses.

Principle of PCR

A single-stranded DNA template is used as a scaffold for the DNA polymerase enzyme, which guides the synthesis of DNA from deoxynucleotide substrates. When a specially created oligonucleotide is annealed to a longer template DNA. DNA polymerase adds nucleotides to the 3′ end of the molecule. Thus, DNA polymerase can employ a synthetic oligonucleotide as a primer and extend its 3′ end to create an extended stretch of double-stranded DNA when annealed to a single-stranded template with a region complementary to the oligonucleotide.

Parts of a PCR Machine

• Thermocycler has a thermal block with holes where tubes holding the reaction mixtures can be inserted.
• It comes with a heated lid pressed against the reaction tube lids. The lid prevents condensation of water from the reaction mixtures on the insides of the lids.
• Rotation of the lid knob clockwise lowers the heating plate that pressurizes the cap of the tubes to sit firmly in the block and ensure better contact. Conversely, its rotation in counter-clockwise directions pops up the lid so that users can slide the lid to the rear.
• A control panel includes a large graphical display that facilitates easy reading and displays the current status of the various system features and functions and a Key-Pad used to enter the various protocols and settings.
• The presence of air vents in the front, lateral and bottom sides facilitates air output and air intake.

PCR Components (PCR reagents)

DNA Template

• It is crucial to choose the appropriate templates for PCR. 
• RNA templates are needed to create complementary DNA in RT-qPCR whereas DNA templates are needed for traditional PCR.
• Consequently, the first stage of a successful PCR requires the use of a reliable PCR template preparation kit.

Thermostable DNA polymerase

• Since the initial step of PCR requires separating DNA strands at a high temperature (90 °C), all PCR reactions require a DNA polymerase that can function at a high temperature (about 70 °C).
• A popular DNA polymerase for PCR known as Taq polymerase, obtained from the thermophilic bacteria Thermus aquaticus is a heat-stable enzyme.

Oligonucleotide Primers

• Primers are short strands of nucleotides (DNA or RNA) that are complementary to the template DNA and act as a starting point for the DNA/RNA polymerase to begin synthesizing new DNA. 
• Primers are short strands of nucleotides (DNA or RNA) that are complementary to the template DNA and act as the DNA/RNA polymerase’s starting point for DNA synthesis. They are necessary for the start of DNA synthesis. Lower temperatures (50–65°C) are needed for annealing primers to single-strand DNA than for denaturation which generates hydrogen bonds after the annealing process is finished.

Deoxyribonucleotide triphosphate(dNTPs)

• dNTPs are required for DNA polymerase to be able to synthesize DNA.

Buffer System

• The best conditions are always maintained for the PCR reaction thanks to PCR buffers. 
• Tris-HCl, potassium chloride (KCl), and magnesium chloride (MgCl₂) make up the majority of the ingredients in the PCR buffer.
• In order to keep the pH steady during PCR, tris-HCl and KCl are used.  In order to ensure that DNA polymerase performs properly DNA synthesis during PCR, magnesium ions serve as cofactors for the enzyme.

Steps of PCR

Denaturation

• When the reaction mixture is heated for 0.5 to 2 minutes to 94°C, denaturation takes place.
• A single-stranded DNA is created as a result of the hydrogen bonds between the DNA’s two strands being broken.
• The single DNA strands now serve as a template for the synthesis of additional DNA strands.

Annealing

• For around 20 to 40 seconds, the reaction temperature is decreased to 54 to 60 °C.
• The primers attach to the template DNA’s complimentary sequences in this circumstance. 
• Primer sequences are 20–30 bases long, single-strand DNA or RNA segments.
• They act as the precursor in the production of DNA.
• There are two primers—a forward primer and a reverse primer—the two separated strands run in opposing directions.

Elongation

• The temperature is increased to between 72 and 80 degrees Celsius at this stage. 
• The Taq polymerase enzyme tacks the bases onto the primer’s 3′ end. As a result, the DNA stretches from 5′ to 3′.
• Taq Polymerase can withstand extremely high temperatures. A double-stranded DNA molecule is produced as a result.

In order to obtain several DNA sequences of interest in a short amount of time, these three procedures are done 20–40 times.

PCR Machine Operating Procedure

• Initially, the sample is heated with the use of a PCR machine which causes the denaturation and separation of the DNA into two pieces of single-stranded DNA.
• Following that, an enzyme known as “Taq polymerase” creates or constructs two new DNA strands using the old strands as templates.
• Due to this procedure, the original DNA is duplicated, with one old and one new strand of DNA present in each new molecule. 
• After that, each of these strands can e used to make two other copies, and so on. 
• There are more than one billion exact replicas of the original DNA segment when the cycle of denaturing and creating new DNA is repeated up to 30 or 40 times.
• The entire PCR cycling procedure is automated and can be finished in a matter of hours. The reaction is controlled by a device known as a thermocycler, which is configured to change the temperature of the reaction every few minutes to enable DNA denaturation and synthesis.

Types of PCR

Real-time PCR, also known as quantitative PCR or qPCR, is a technique for monitoring and quantifying PCR results in real-time by labeling DNA molecules with fluorescent dye.

Reverse-Transcriptase (RT-PCR) converts RNA to DNA in the process of producing complementary DNA (cDNA).

Nested PCR lowers the possibility of undesired products by conducting a second PCR using new primers “nested” within the initial 25–35 PCR cycles.

Hot Start PCR uses heat to denature antibodies that are used to inactivate Taq polymerase.

Multiplex PCR multiplies several fragments in a single DNA sample by using a number of primers.

Long-range PCR uses a variety of polymerases, to generate larger DNA ranges.

In situ PCR is a type of PCR that occurs in cells or fixed tissue on a slide

One strand of the target DNA is amplified by an asymmetric PCR process.

Use of overlapping primers in assembly PCR allows for the amplification of longer DNA fragments.

PCR Applications

1. Gene transcription

• PCR can investigate differences in gene transcription among various cell types, tissues, and species at a particular time period.
• Reverse transcription is used to create cDNA by isolating RNA  from samples of interest.
• The quantity of cDNA produced by PCR can then be used to calculate the original RNA levels for a particular gene.

2. Genotyping

• Sequence differences in alleles of certain cells or organisms can be determined.
• Genotyping of transgenic organisms facilitates the amplification of the mutation or a transgenic part.

3. Cloning and mutagenesis

• PCR cloning makes it possible to breed new strains of bacteria with altered genetic makeup via the insertion of amplified dsDNA fragments into the vectors such as gDNA, cDNA, and plasmid DNA.
• Cloning assists in introducing point mutations using site-directed mutagenesis which itself employs the recombinant PCR method.
• It also helps to create novel gene fusions.

4. Sequencing

• Sequencing accompanies the amplification of template DNA, their purification, and processing through a sequencing step.
• PCR is also employed in next-generation sequencing (NGS) during the library preparation phase to quantify DNA samples and tag them with sequencing adaptors for multiplexing.

5. Medicine and biomedical research

• Medical applications include genetic changes linked to disease to identifying infectious organisms. Prenatal genetic testing employs PCR to detect chromosomal abnormalities and genetic mutations in the pregnancy, providing expecting parents with crucial information about the likelihood that their children will have a particular genetic illness.
• It can also be employed as a preimplantation genetic diagnosis technique to screen embryos for in vitro fertilization (IVF).

6. Forensic Science

• PCR can be useful for forensic investigations to identify the sources of samples and paternity testing.
• It is used in molecular archaeology to amplify DNA from artifacts.

7. Environmental microbiology and food safety

• Pathogens can be found using PCR, not just in patient samples but also in matrices like food and water. This is important for both diagnosing and preventing infectious diseases.

PCR Advantages

• Allows for quicker, more informed decision making
• Rapid identification of bacteremia, especially for specimens with low bacterial counts
• Effective in identifying cases in extrapulmonary specimens that smear and/or culture may have missed.
• Important for identifying certain diseases that are challenging to cultivate in vitro or take a long time to cultivate. Significantly faster at producing results than cultivating
• It is still regarded as an auxiliary test for some diagnostic procedures that depend on smear and culture such as Tuberculosis.
• It has the ability to test for antimicrobial resistance.

PCR Limitations

• Unknown target amplification is not possible with PCR. The design of the primers requires knowledge about the target sequence in advance.
• Error-prone DNA polymerases have the potential to lead to PCR product mutations.
• PCR is extremely susceptible to contamination. Results could be misinterpreted or confusing if there is even a little amount of contaminated DNA.
• With the increase in amplicon size, PCR efficiency decreases.

Precautions using PCR Machine

• Only tubes and plates designed for the PCR machine should be used.
• Before running, double-check tubes and, in particular, plates are tightly sealed.
• Solution spills should be cleaned up, and biohazard containers should be used for disposal.
• Be cautious when using a PCR machine lid. If you drop or bang lids, this could get broken.
• Once, the use of the PCR machine is finished, it should be turned off.
• Prior to beginning a run, make sure the PCR heating block is clean. Before beginning, inspect every tube holder.
• To ensure that the lid sits flat against the top of the tubes for even heating and sealing, distribute the tubers evenly over the block.

PCR Machine Examples

Biometra TAdvanced Thermal Cycler Series (Analytik Jena)

• Twelve distinct sample blocks, including a high-end 96-well silver block
• With today’s powerful control electronics, even an aluminum block can produce faster heating and cooling than is often possible with silver block-only instruments.
• Fastest Ramping, Highest Accuracy, and Intelligent Block/Well Control (RAC) provide the highest temperature uniformity and reproducibility with zero over- or undershoot of the programmed target temperature.
• Fast heating, continuous contact pressure, and simplicity of use are all features of High-Performance Smart Lid Technology (HPSL).

MiniAmp™ Plus Thermal Cycler (ThermoFisher Scientific)

• VeriFlex Blocks have three distinct temperature zones that allow you to precisely manage the temperature for your PCR optimization.
• A 5-inch intuitive color touch-screen that is simple to program and instruct new users on Compact size—With a footprint of just 19 cm wide and 39 cm deep, the MiniAmp Thermal Cycler can fit almost any place. Thermo Fisher Connect’s WiFi capabilities enable you to design and securely upload your methods from any mobile device or desktop computer.
•  Additionally, because its airflow is front to back, you can place multiple unit side by side to save valuable bench space.

PCR Thermal Cyclers (Esco)

• For all types of PCR processes, including gradient PCR, touchdown PCR, high throughput PCR, in situ PCR, and others that use a range of PCR tubes, strips, plates, and slides, 
• Esco offers a selection of conventional thermal cycler and real time thermal cycler models that are built to meet stringent requirements.
•  In order to establish and maintain accurate control and quick ramp rates with little overshoot or undershoot for process speed and accuracy, Swift thermal cyclers use cutting-edge Peltier temperature control technology.

GET-S SERIES THERMAL CYCLER (Bio-gener)

• An extended service life peltier is used in the GET-S series thermal cycler. Its maximum ramping rate is 4.5 °C/s, and its cycle times exceed one million. 
• The product integrates a number of cutting-edge technology, including the Android operating system, a color capacitive touch screen, many block possibilities, an integrated WIFI module, PC software control functionality, email notification functionality, a large storage capacity, and more. 
• The aforementioned features enable PCR’s great performance and satisfy more demanding experiment requirements.

MiniAmp Plus Thermal Cycler and the MiniAmp Thermal Cycler (Delta Science)

• Fits on any benchtop because to its small size.
• With Thermo Fisher Connect, you can easily access your instrument from any location that has access to the internet.
• When you upgrade to the Applied Biosystems MiniAmpTM Plus Thermal Cycler, you have the option to get VeriFlexTM temperature control technology for PCR optimization.

References

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2. Al-Mohanna, Moshtaq & International, Labnet. (2017). Labnet MultiGene Manual. 10.13140/RG.2.2.23499.80164.
3. Jain, A., Jain, R., Jain, S. (2020). Polymerase Chain Reaction. In: Basic Techniques in Biochemistry, Microbiology and Molecular Biology. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9861-6_14
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