Polymerase chain reaction (PCR) is a powerful molecular biology technique used to amplify DNA. It was first developed by Kary Mullis in 1983 and has since revolutionized the field of molecular biology. The process involves four main steps: denaturation, annealing, extension, and repeat. In this article, we will discuss each step in detail.
Step 1: Denaturation
The first step of PCR is denaturation, which involves heating the DNA to separate the two strands. The double-stranded DNA is heated to a temperature of around 94°C to 96°C, causing the hydrogen bonds between the complementary base pairs to break, resulting in two separate strands.
The purpose of denaturation is to create single-stranded DNA templates for the next step of PCR, annealing.
Step 2: Annealing
The second step is annealing, which involves cooling the DNA to a temperature between 50°C to 60°C. During this step, the primers bind to their complementary sequences on the single-stranded DNA. The primers are short pieces of DNA that are complementary to the sequences at the ends of the region of interest.
The purpose of annealing is to allow the primers to hybridize, or bind, to the single-stranded DNA template, specifically to the regions flanking the target sequence. The annealing temperature is chosen based on the melting temperature of the primers and the DNA template.
Step 3: Extension
The third step is extension, which involves heating the DNA to a temperature between 72°C to 74°C. During this step, the DNA polymerase enzyme adds nucleotides to the 3' end of the primers, synthesizing new strands of DNA. The polymerase extends the primers in a 5' to 3' direction, adding nucleotides to the growing DNA strand.
The purpose of extension is to synthesize a new DNA strand complementary to the single-stranded template using the primers as a starting point. Taq polymerase, a heat-stable DNA polymerase, is commonly used in PCR because it is not denatured by the high temperatures used in denaturation.
Step 4: Repeat
After the first cycle of denaturation, annealing, and extension, the process is repeated multiple times. Each cycle doubles the number of copies of the target DNA sequence. The number of cycles depends on the amount of starting DNA, the length of the target DNA sequence, and the efficiency of the reaction.
The number of cycles is typically between 20 and 40, and each cycle takes between 30 seconds to 2 minutes, depending on the equipment and reagents used.
The final product of PCR is a large number of copies of the target DNA sequence, which can be analyzed by various techniques, such as gel electrophoresis, sequencing, or cloning.
In conclusion, PCR is a widely used technique in molecular biology, and the four steps of denaturation, annealing, extension, and repeat are crucial for successful amplification of DNA. The technique has revolutionized the field of genetics and has applications in medical diagnostics, forensics, and biotechnology.
