How does PCR work?
PCR (Polymerase Chain Reaction) is a technique used in molecular biology to amplify a specific region of DNA, making millions of copies of it. The basis of PCR was discovered by Kary B. Mullis in 1985 via the understanding that small quantity of DNA to be replicated rapidly and in large amounts within a brief timeframe. Today, the technique is widely used in research, clinical diagnostics, and forensics. Here are the main steps involved in PCR:
Denaturation: The first step of PCR is denaturation, which involves heating the DNA sample to a high temperature (usually around 95°C) for a short period of time (usually 30 seconds to 1 minute). This breaks the hydrogen bonds between the two strands of the DNA molecule, separating them and creating two single-stranded DNA templates.
Annealing: The next step is annealing, where the temperature is lowered to around 50-60°C for a short period of time (usually 30 seconds to 1 minute). During this step, short pieces of DNA called primers are added to the reaction mixture. These primers are designed to bind specifically to the single-stranded DNA templates at either end of the target sequence that we want to amplify.
Extension: The third step is extension, where the temperature is raised to around 72°C for a longer period of time (usually 1-2 minutes). During this step, a DNA polymerase enzyme adds nucleotides (the building blocks of DNA) to the primers, extending them along the template strands in a 5' to 3' direction. As the polymerase moves along the template strands, it creates a complimentary copy of the target sequence, doubling the amount of DNA.
Repeat: After the extension step, the cycle is repeated from step one, starting with the denaturation step. The whole process is typically repeated 20-40 times, each cycle doubling the amount of DNA and generating millions of copies of the target sequence.
Reverse Transcriptase - PCR
RT-PCR is similar to PCR, but it involves the additional step of reverse transcription to convert RNA into complementary DNA (cDNA) before amplification. This is necessary because PCR can only amplify DNA, not RNA. RT-PCR is commonly used to detect and quantify gene expression by measuring the amount of cDNA produced from RNA samples.
qPCR is a variation of PCR that uses fluorescent probes to detect the amount of DNA or cDNA produced in real-time during the amplification process. qPCR is more quantitative than regular PCR or RT-PCR because it can measure the amount of DNA or cDNA present in a sample at the beginning of the reaction and at the end. This makes qPCR useful for quantifying gene expression, detecting genetic mutations, and quantifying viral loads.
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