Polymerase Chain Reaction (PCR) is a powerful technique used in molecular biology to amplify a specific DNA sequence. This method has revolutionized various fields such as genetics, forensics, and medical research. In this blog post, we will provide a detailed explanation of the procedure of Polymerase Chain Reaction.
The first step in the PCR process is denaturation. The DNA sample containing the target sequence is heated to a high temperature, typically around 95°C. This causes the double-stranded DNA to separate into two single strands. Denaturation is crucial as it allows the DNA to be accessible for the following steps.
After denaturation, the temperature is lowered to around 50-65°C. In this step, short DNA primers are added to the DNA sample. These primers are designed to bind specifically to the sequences flanking the target DNA region. By binding to the complementary sequences, the primers provide a starting point for the DNA polymerase to initiate replication.
Once the primers are bound, the temperature is raised to around 72°C, which is the optimal temperature for DNA polymerase activity. DNA polymerase, a key enzyme in PCR, synthesizes new DNA strands by adding nucleotides to the primers. This process extends the DNA sequence, creating two new strands that are complementary to the original template.
4. Repeat Cycle:
The denaturation, annealing, and extension steps are repeated multiple times in a PCR machine. Each cycle doubles the amount of DNA, resulting in exponential amplification of the target sequence. The number of cycles depends on the initial amount of DNA and the desired final quantity.
5. Final Extension:
After the desired number of cycles, a final extension step is performed at a slightly higher temperature, typically around 72°C. This ensures that any remaining incomplete DNA strands are fully extended.
Finally, the temperature is lowered to around 4-15°C to stop the PCR reaction. The amplified DNA can now be used for various applications, such as DNA sequencing, cloning, or further analysis.
PCR has revolutionized molecular biology by enabling the amplification of specific DNA sequences. It has become an essential tool in various fields, including medical diagnostics, genetic research, and forensic analysis. Understanding the procedure of PCR is crucial for researchers and scientists working with DNA.
Overall, the Polymerase Chain Reaction (PCR) procedure involves denaturation, annealing, extension, and repeating cycles to amplify a specific DNA sequence. This technique has greatly advanced our understanding of genetics and has numerous practical applications in various scientific disciplines.