13/04/2023
Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify a small amount of DNA into a larger amount, making it easier to study and analyze. The technique uses a DNA polymerase enzyme to replicate specific segments of DNA many times over, producing millions of copies of a specific DNA sequence.
PCR involves three main steps:
1. Denaturation: The double-stranded DNA is heated to a high temperature, causing the two strands to separate (denature) into single strands.
2. Annealing: The temperature is lowered, allowing short pieces of DNA called primers to attach (anneal) to the DNA strands at specific locations.
3. Extension: The temperature is raised again, and the polymerase enzyme extends the primers by adding new nucleotides to the DNA strand, creating a copy of the original sequence. This process is repeated several times, with each cycle resulting in an exponential increase in the number of copies of the targeted DNA sequence.
Types of PCR
1. Standard PCR (Polymerase Chain Reaction): The standard PCR amplifies a DNA fragment by repeated cycles of denaturation, annealing, and extension.
2. Nested PCR: Nested PCR is a modification of standard PCR, which increases the specificity and sensitivity of the reaction by performing two successive PCR amplifications, using two sets of primer pairs designed to anneal to different regions of the target DNA.
3. Reverse Transcription PCR (RT-PCR): RT-PCR is used to amplify RNA molecules by converting them into complementary DNA (cDNA) using reverse transcriptase enzyme before the PCR amplification step.
4. Real-time PCR: Real-time PCR is a quantitative PCR technique that monitors the amplification of DNA in real-time, allowing the measurement of the amount of DNA present during each cycle of PCR amplification.
5. Multiplex PCR: Multiplex PCR amplifies multiple target DNA sequences simultaneously in a single reaction using multiple primer sets.
6. Inverse PCR: Inverse PCR is a technique that amplifies a DNA fragment whose sequence is known, but whose flanking regions are unknown by using primers that anneal to the known sequence and then amplify outwards.
7. Touchdown PCR: Touchdown PCR is a technique that starts with a high annealing temperature and gradually decreases the temperature with each cycle, thereby increasing the specificity of the reaction.
8. Hot-start PCR: Hot-start PCR is a technique that reduces non-specific amplification by preventing DNA polymerase activity until a certain temperature is reached, usually above 70°C.
9. Colony PCR: Colony PCR is a quick and cost-effective method used to screen bacterial colonies for the presence of a desired DNA fragment by directly amplifying the DNA from a colony.
10. Digital PCR: Digital PCR is a new technique that enables the absolute quantification of DNA targets in a sample, by partitioning the sample into thousands of individual PCR reactions and then counting the number of positive reactions.
PCR is a widely used tool in many areas of biological research, including genetics, disease diagnosis, forensic science, and evolutionary biology.
