Polymerase chain reaction (PCR) is a 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. PCR allows the generation of large quantities of a specific DNA sequence from a small amount of starting material. It is used in many applications, such as medical diagnostics, forensics, and biotechnology.
The basic principles of PCR involve the use of a DNA polymerase enzyme, primers, and nucleotides to amplify a specific region of DNA. The process involves four main steps: denaturation, annealing, extension, and repeat.
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 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.
PCR has many applications in molecular biology, including medical diagnostics, such as the detection of viral infections and genetic diseases, forensic analysis, and biotechnology, such as genetic engineering and gene expression analysis.
In conclusion, PCR is a powerful technique in molecular biology that allows for the amplification of DNA. The technique has revolutionized the field of genetics and has many applications in various fields of science.