How PCR works : A Step-by-Step Guide to Polymerase Chain Reaction (PCR)

What is  Polymerase Chain Reaction (PCR):

Polymerase Chain Reaction (PCR) is a molecular biology lab technique that creates millions to billions of copies of a specific section of DNA or gene of interest. This procedure enables the examination of genetic material, allowing for a variety of uses in research, diagnostics, and biotechnology. Kary Mullis (December 28, 1944 – August 7, 2019) was an American biochemist who created PCR in 1983. A thermostable DNA polymerase and a DNA primer designed for the DNA template are required for PCR. The reaction cycle is repeated many times in the polymerase chain reaction to produce billions of copies.

How Does PCR Work?

PCR works in a temperature cycle. The three key steps of PCR are denaturation, annealing, and extension.

1. Denaturation:

The DNA template is heated (94°C-98°C) during this initial step, causing the double-stranded DNA to split into two single strands. It disrupts the hydrogen bonds that exist between base pairs. Denaturation causes to development of single strands of DNA.

2. Annealing:

The reaction temperature decreases (55°C-70°C) to allow the PCR primers to bind to the ssDNA. Primers are small single strands of nucleic acid. Two sets of primers are designed to bind to complementary sequences at the 3' end of each strand. Primer design is critical to the effectiveness of the PCR process. If the primer sets are poorly constructed, the PCR may fail because no copies are produced or mismatches occur.

3. Extension:

DNA polymerase uses the primers as starting points to create a new strand of DNA that is complementary to the DNA template. Raise the reaction temperatures to (72°C-80°C) so that Taq polymerase can stretch the primers and synthesize new DNA strands. This cycle repeated several times, increasing the targeted DNA region exponentially.

Types of PCR:

There are several PCR variants designed for specific applications:

1. Conventional PCR: Standard PCR for DNA amplification.
2. Quantitative PCR (qPCR): Measures the amount of DNA in real-time, allowing for quantitative analysis.
3. Reverse Transcription PCR (RT-PCR): Converts RNA into complementary DNA before amplification.
4. Nested PCR: Uses two sets of primers in sequential reactions for increased specificity.
5. Multiplex PCR: Amplifies multiple DNA targets in a single reaction.
6. Digital PCR: Quantifies DNA by distributing the sample into thousands of individual reactions.

DNA Extraction Methods for PCR:

Before PCR, DNA must be extracted from cells. Common methods include:

1. Phenol-Chloroform Extraction: Utilizes organic solvents to separate DNA from proteins.
2. Silica-Membrane Spin Columns: Binds DNA to a silica matrix, allowing for purification. It can be also done, Kit.
3. Chelex Resin Method: Uses chelating resin to bind metal ions, facilitating DNA release.

Taq Polymerase:

Taq polymerase is a heat-stable enzyme derived from the bacterium Thermus aquaticus. It withstands the high temperatures of denaturation in PCR, making it an essential component of the reaction.

PCR Primers:

PCR primers are short, single-stranded DNA sequences that bind to complementary sequences on the target DNA, providing a starting point for DNA synthesis.

Taq Polymerase vs. Primer:

• Polymerase: Enzyme responsible for synthesizing new DNA strands during extension.
• Primer: Short DNA sequence that initiates DNA synthesis by providing a starting point for the polymerase.

Thermocycler Machine:

The thermocycler is a specialized machine controlling temperature variations during the PCR process. It automates the repeated heating and cooling cycles required for denaturation, annealing, and extension. You can set temperature cycles according to your needs and your specific protocol.

Visualization by Gel Electrophoresis:

PCR is used to amplify specific DNA regions while gel electrophoresis is used to view and characterize the amplified results. The PCR products are loaded onto an agarose gel and exposed to an electric field, causing the DNA fragments to migrate through the gel according to size. To scale the bands, a PCR ladder was also loaded on the gel. Smaller pieces move faster through the gel matrix than larger fragments.  Following electrophoresis, the gel is dyed with a DNA-binding dye, revealing discrete bands representing different-sized DNA fragments visible under UV illumination. This enables researchers to check the PCR reaction's success, determine the size of the amplified fragments, and evaluate the purity of the PCR result.

Gel image

PCR Protocol:

Remember this is the general protocol for the idea  always follow your specific test protocol and supplier guidelines

A standard Polymerase Chain Reaction (PCR) procedure involves four key steps. Firstly, PCR tubes are filled with the necessary chemicals, including the master mix and template DNA. Secondly, the components are combined and subjected to centrifugation to ensure thorough mixing and settling of contents. The third step involves the amplification process, where the prepared mixture is cycled through a thermocycler following specific settings for temperature and primer sequences. Finally, the amplified DNA is analyzed in the fourth step using agarose gel electrophoresis, and the results are visualized through ethidium bromide staining. The Polymerase Chain Reaction (PCR) is a widely used molecular biology technique for DNA amplification. A typical PCR reaction, with a total volume of 20 to 50 μl, includes various components. Genomic DNA or cDNA, ranging from 0.1 to 1 μg (with approximately 0.1 μg sufficient for plasmid DNA), is combined with a 10X PCR buffer to achieve a final concentration of 1X. A 4 mM dNTP mix, containing dCTP, dATP, dGTP, and dTTP, is added to reach a final concentration of 0.2 mM. Both forward and reverse primers are introduced at a final concentration of 0.1 μM to 1 μM for each primer, along with 1 unit/μl of Taq polymerase. The volume is adjusted with DNA and DNase-free water to complete the reaction. For instance, a 20 μl reaction includes 1 μl dsDNA template, 2 μl 10X buffer, 1 μl 4 mM dNTP mix, 1 μl 10 μM forward primer, 1 μl 10 μM reverse primer, 1 μl 1 unit/μl Taq polymerase, and 13 μl water. The reagents are combined, kept on ice, and later loaded into a thermocycler for amplification. Post-PCR, samples are held at 4°C, and the DNA is prepared for gel electrophoresis by adding 1/10 volume stop-loading buffer. The analysis includes the use of size markers in at least one well on the gel to assess the amplified DNA.

Basic Applications of PCR:

•  DNA Cloning: The process of amplifying DNA for insertion into vectors.
•  Genetic Testing: This involves the detection of genetic disorders or predispositions.
•  Forensic Analysis: The use of DNA profiling in criminal investigations.
•  Pathogen Detection: Identifying infectious agents most recent example is COVID-19.
•  Environmental Monitoring: Microorganism detection in environmental samples.
•  Mutation Analysis: Finding genetic mutations.
•  Expression Profiling: Measuring gene expression levels.
•  Pharmacogenetics: The study of genetic variables that influence medication reactions.
•  DNA Sequencing: The preparation of DNA templates for sequencing.
• Ancient DNA Studies: Amplification of degraded DNA from historical or archeological sources.