Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify DNA. The process involves four basic steps: denaturation, annealing, extension, and repeat. In this article, we will discuss these steps 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.
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.
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.
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 four steps of PCR are essential for amplifying DNA and generating enough copies for downstream applications, such as sequencing or cloning. By automating the process, PCR has become an essential tool in molecular biology and has revolutionized the field of genetics.