History and Advancements:

The Polymerase Chain Reaction, pioneered by Kary B. Mullis in the 1980s, revolutionized the way scientists study DNA. This Nobel Prize-winning innovation allowed for the exponential amplification of DNA sequences, making it an invaluable tool in various scientific disciplines.

Over the years, PCR technology has undergone significant advancements. From the introduction of hot-start PCR to the development of high-fidelity polymerases, the technique has become more robust, efficient, and versatile. Real-Time PCR, a more recent innovation, brought quantitative capabilities to the table, enabling precise measurements of DNA amplification during the process.

Conventional PCR vs. Real-Time PCR:

Real-Time PCR: Real-Time PCR, also known as quantitative PCR (qPCR), revolutionized the field by allowing scientists to monitor the DNA amplification process in real-time. This technique utilizes fluorescent dyes or probes to track the accumulation of PCR products, providing accurate and quantitative data.

Conventional PCR, the original method, involves repeated cycles of heating and cooling, resulting in the exponential amplification of the target DNA sequence. However, this method provides limited information about the amplification process. In contrast, real-time PCR, a more sophisticated variant, utilizes fluorescent probes that emit light as the target DNA is amplified. This real-time monitoring allows for precise quantification of DNA and the detection of mutations or polymorphisms.

Conventional PCR: Conventional PCR involves three main steps: denaturation, annealing, and extension. The process is repeated in cycles, with each cycle doubling the amount of target DNA. While effective, conventional PCR lacks the ability to provide real-time data on the DNA amplification process.

Advantages and Disadvantages:

Advantages:

1. Quantitative Data: Real-Time PCR allows for the quantification of initial DNA amounts, providing precise information on the starting material.
2. High Sensitivity: Real-Time PCR can detect low amounts of DNA, making it suitable for applications such as gene expression analysis.
3. Time Efficiency: Real-Time PCR eliminates the need for gel electrophoresis, reducing overall processing time.

Disadvantages:

1. Cost: Real-Time PCR instruments and reagents can be more expensive than those used in conventional PCR.
2. Complexity: Real-Time PCR requires specialized equipment and expertise, making it less accessible for some researchers.

The Key components of PCR mastermix

• Template DNA: The DNA molecule that contains the target sequence to be amplified.

• Primers: Short DNA sequences that are complementary to specific regions of the target DNA. Primers serve as starting points for DNA synthesis.

• DNA polymerase: An enzyme that catalyzes the synthesis of new DNA strands. Taq polymerase, isolated from the bacterium Thermus aquaticus, is commonly used in PCR due to its thermostability.

• Deoxynucleoside triphosphates (dNTPs): The building blocks of new DNA strands. dNTPs consist of the four bases: adenine (A), thymine (T), guanine (G), and cytosine (C).

• Buffer: A solution that provides the appropriate pH and ionic conditions for DNA polymerase to function optimally.

• Magnesium ions (Mg2+): A cofactor required for DNA polymerase activity.

The basic steps of PCR are:

1. Denaturation: The double-stranded DNA is heated to separate the two strands.

2. Annealing: Primers bind to their complementary sequences on the single-stranded DNA.

3. Extension: DNA polymerase synthesizes new complementary DNA strands using the primers as starting points.

4. Cycles: Steps 1-3 are repeated for 20-30 cycles, resulting in the exponential amplification of the target DNA sequence.

Now let us see how to setup a PCR reaction and what are all the materials required for setting the PCR and the steps to follow while setting up the PCR:

Materials Required:

• PCR tubes or microplates
• Template DNA
• Primers (forward and reverse)
• Probe (incase of realtime PCR)
• DNA polymerase
• dNTPs
• PCR buffer
• MgCl2 solution
• Mineral oil (optional)
• Thermal cycler

Procedure:

Prepare the master mix:

a. In a clean microfuge tube, combine the following reagents:

• PCR buffer
• MgCl2 solution
• dNTPs
• DNA polymerase

b. Mix gently by pipetting up and down. c. Keep the master mix on ice until ready to use.

Prepare the reaction mix:

a. In a separate clean microfuge tube, combine the following reagents:

• Master mix
• Template DNA
• Primers (forward and reverse)

b. Mix gently by pipetting up and down.

c. If using mineral oil, add a small amount to the top of the reaction mix to prevent evaporation.

Transfer the reaction mix to PCR tubes or microplates:

a. Carefully transfer the reaction mix to PCR tubes or microplates, ensuring that each tube or well receives the same amount of mix.

b. Close the tubes or seal the plates tightly to prevent contamination.

Place the PCR tubes or microplates in the thermal cycler:

a. Program the thermal cycler with the appropriate PCR protocol.

b. The PCR protocol will typically consist of 30-40 cycles of the following steps:

• Denaturation: 95°C for 30 seconds
• Annealing: 55-65°C for 30 seconds
• Extension: 72°C for 30 seconds

The annealing temperature is decided based on the Tm of the primers.

Run the PCR program:

a. Start the PCR program and let the thermal cycler run its course.

b. The PCR program will typically take 1-2 hours to complete (depending on the cycling conditions)

Analyze the PCR products:

a. Once the PCR program is complete, remove the PCR tubes or microplates from the thermal cycler.

b. Analyze the PCR products using an appropriate method, such as agarose gel electrophoresis.

If your reaction is based on probe, real-time application can be viewed.

Additional tips:

• Always use sterile technique when setting up PCR reactions to prevent contamination.
• Use high-quality reagents and primers to ensure optimal results.
• Optimize the annealing temperature and primer concentration for your specific primers and template DNA.
• Use negative and positive controls to ensure the accuracy of your results.

New Developments:

The field of PCR is constantly evolving, with researchers exploring new techniques and technologies. Recent developments include the introduction of digital PCR, a method that partitions DNA samples into thousands of individual reactions, enhancing precision and sensitivity. Additionally, isothermal amplification techniques have emerged as alternatives to traditional PCR, simplifying the amplification process.

PCR is being integrated with next-generation sequencing technologies, facilitating the analysis of large genomic regions.

Applications of Real-Time PCR:

Real-Time PCR finds applications across diverse scientific domains, including:

1. Gene Expression Analysis: Quantifying the expression levels of specific genes in various tissues or under different conditions.
2. Pathogen Detection: Identifying and quantifying pathogens in clinical samples for diagnostic purposes.
3. Genetic Variation Analysis: Investigating single nucleotide polymorphisms (SNPs) and mutations associated with diseases.
4. Environmental Monitoring: Detecting and quantifying microbial contaminants in environmental samples.
5. Forensic Science: Analyzing DNA samples for identification and criminal investigations.

Polymerase Chain Reaction and Real-Time PCR have undoubtedly reshaped the landscape of molecular biology.