Introduction
In the field of molecular biology, the polymerase chain reaction (PCR) is a widely used technique to amplify specific DNA sequences. It is a crucial tool in various applications, including genetic research, diagnostics, and forensic analysis. PCR involves a series of temperature-dependent steps that facilitate the synthesis of complementary DNA strands.
Denaturation
The first step in PCR is denaturation, where the DNA template is heated to a high temperature (typically 94-98°C). This causes the double-stranded DNA to separate into two individual strands, breaking the hydrogen bonds between the complementary bases. Denaturation results in the formation of single-stranded DNA, which serves as a template for subsequent steps.
Annealing
Following denaturation, the temperature is lowered to allow the DNA primers to anneal to the single-stranded template. Primers are short DNA sequences that are complementary to the regions flanking the target DNA sequence. They provide a starting point for DNA synthesis. During annealing, the primers bind to their complementary sequences on the template DNA, forming stable primer-template complexes.
Extension/Elongation
The next step is extension or elongation, where the temperature is increased to the optimal range for DNA polymerase activity (typically 72°C). DNA polymerase, a key enzyme in PCR, synthesizes new DNA strands by adding complementary nucleotides to the primers. It reads the template DNA and adds nucleotides in a complementary manner, following the rules of base pairing (A with T, and G with C).
Complementary DNA Strand Synthesis
The extension step is the critical stage where complementary DNA strands are synthesized. As DNA polymerase moves along the template DNA, it incorporates nucleotides that are complementary to the template strand. For example, if the template strand has an A base, DNA polymerase adds a T nucleotide to the growing complementary strand. This process continues until the entire template DNA is replicated, resulting in the synthesis of two complete complementary DNA strands.
Role of DNA Polymerase
DNA polymerase is the key enzyme responsible for the synthesis of complementary DNA strands during PCR. It has a unique ability to read the template DNA and selectively incorporate the corresponding nucleotides. DNA polymerase also possesses proofreading capabilities, ensuring the accuracy of DNA replication by correcting any errors that may occur during synthesis.
Multiple Cycles
PCR typically consists of multiple cycles of denaturation, annealing, and extension. Each cycle doubles the amount of DNA, resulting in an exponential amplification of the target sequence. The number of cycles performed during PCR can vary depending on the desired amount of DNA and the sensitivity of the detection method.
Conclusion
In summary, the extension or elongation step in PCR is responsible for synthesizing complementary DNA strands. DNA polymerase, the key enzyme in PCR, reads the template DNA and adds nucleotides in a complementary manner, resulting in the replication of the target DNA sequence. Understanding the steps involved in PCR is crucial for successful amplification and analysis of specific DNA sequences.
Sources:
1. Mullis, K., & Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335-350.
2. Saiki, R. K., et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239(4839), 487-491.