Polymerase Chain Reaction (PCR) is a nucleic acid amplification technique used to replicate DNA or RNA in vitro enzymatically. It rapidly produces millions of copies of a specific DNA segment, earning it the nickname “molecular photocopying”.
- Principle: PCR relies on temperature-dependent enzymatic cycles to amplify DNA or RNA.
- Non-Culture-Based: It doesn’t require culturing cells; instead, it directly amplifies nucleic acids.
- Cyclic Process: Through multiple cycles of denaturation, annealing, and extension, PCR quickly generates billions of copies of the target DNA or RNA segment.
- Hybridization and Replication: PCR combines nucleic acid hybridization and replication principles. It starts by denaturing double-stranded DNA, then selects a segment for amplification using specific primers, followed by replication with DNA polymerase.
- Origin: PCR was developed in the mid-1980s by Kary Mullis and his team. Mullis and Michael Smith were awarded the Nobel Prize in Chemistry in 1993 for this groundbreaking innovation.
- Revolutionary Impact: PCR has become a cornerstone in molecular biology and genetics, enabling DNA and RNA analysis with profound implications for research, diagnostics, and various fields.
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Key Points:
Polymerase Chain Reaction (PCR) is a revolutionary technique in molecular biology that allows for the amplification of a specific DNA sequence. Here are some key points about PCR:
- Amplification: PCR rapidly multiplies a specific DNA sequence.
- Heating and Cooling: It involves cycles of heating to separate DNA strands and cooling to allow primers to bind.
- Targeted: Amplifies a specific region of DNA.
- Enzyme: DNA polymerase enzyme synthesizes new DNA strands.
- Applications: Used in research, diagnostics, forensics, and more.
- Versatility: Can detect pathogens, identify genetic disorders, and analyze DNA samples.
- Speed: Provides results in a matter of hours.
- Sensitivity: Detects even small amounts of DNA.
- Variants: Includes qPCR, RT-PCR, nested PCR, multiplex PCR, and dPCR.
- Revolutionary: Revolutionized molecular biology and genetics.
Principle of PCR
The PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group to add the first nucleotide. DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region of double stranded DNA.
Components of PCR
The PCR reaction requires the following components:
- DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.
- DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C.
- Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.
- Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.
- Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.
Procedure of PCR
All the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in an automated, self-contained thermocycler machine.
- Denaturation :
This step involves heating the reaction mixture to 94°C for 15-30 seconds. During this, the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds. - Annealing :
The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. This allows the primers to bind (anneal) to their complementary sequence in the template DNA. - Elongation :
Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). In this step, the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute.
Types of PCR
Several modification of PCR methods have been developed to enhance the utility of this method in diagnostic settings based on their applications. Some of the common types of PCR are;
- Real-Time PCR
- Nested PCR
- Multiplex PCR
- Quantitative PCR
- Arbitrary Primed PCR
Applications of PCR
- Identification and characterization of infectious agents
- Direct detection of microorganisms in patient specimens
- Identification of microorganisms grown in culture
- Detection of antimicrobial resistance
- Investigation of strain relatedness of pathogen of interest
- Genetic fingerprinting (forensic application/paternity testing)
- Detection of mutation ( investigation of genetic diseases)
- Cloning genes
- PCR sequencing
Possible References Used