polymerase chain reaction (PCR)
A technique for amplifying (copying) DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides.
Polymerase Chain Reaction (PCR) is a powerful molecular biology technique that is used to amplify or generate multiple copies of a specific DNA fragment or a target sequence in vitro. It was first introduced by Kary Mullis in 1983. The PCR technique is widely used in research laboratories, clinical diagnostics, and forensic applications.
The PCR technique involves three main steps: Denaturation, Annealing, and Extension, and requires a template DNA, primers, Taq polymerase, and nucleotides (dNTPs).
Denaturation: The first step of PCR involves denaturation of the double-stranded DNA (template DNA) at high temperature (around 95°C). This step separates or melts the double-stranded DNA into two single-stranded DNA molecules.
Annealing: The second step of PCR involves annealing of primers to the complementary sequence of the single-stranded DNA template. The temperature for this step occurs around 50-65°C. The primers provide the starting point for Taq polymerase to amplify the target sequence.
Extension: The final step of PCR involves DNA synthesis by Taq polymerase, which adds nucleotides to the 3’ end of the primer. The temperature required for this step is about 72°C. Taq polymerase is derived from thermophilic bacteria that can grow at high temperatures.
The three steps (Denaturation, Annealing, and Extension) constitute one PCR cycle, and the number of PCR cycles can be repeated up to 30-40 times. After each cycle, the number of copies of the target sequence doubles. Thus, after a few cycles, there are millions of copies of the target sequence.
In conclusion, PCR is an essential tool that has revolutionized the field of molecular biology. It is a fast, sensitive, and specific method for amplifying DNA and is a cornerstone of many modern molecular biology techniques. The technique has found applications in a diverse range of fields like clinical diagnosis, genetic engineering, forensic science, and investigating infectious diseases.
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