Polymerase chain reaction (PCR) is a widely used technique in molecular biology that allows the selective amplification of a specific DNA fragment. The basic steps involved in the PCR process are as follows:
Denaturation: The DNA sample containing the target sequence is heated to a high temperature (usually around 95°C) to denature the double-stranded DNA into single strands.
- Annealing: The temperature is then lowered to around 50-60°C, allowing primers (short DNA sequences that are complementary to the target sequence) to anneal (bind) to the single-stranded DNA.
- Extension: A heat-stable DNA polymerase enzyme (such as Taq polymerase) adds nucleotides to the 3' end of the primers, thereby extending the new DNA strands.
- Repeated cycles: The process of denaturation, annealing, and extension is repeated many times (usually 20-30 cycles), resulting in exponential amplification of the target DNA sequence.
Selective amplification of a DNA fragment can be achieved by designing primers that are specific to the target sequence. These primers should only anneal to the target sequence and not to any other DNA in the sample. By using appropriate annealing temperatures and PCR conditions, only the target sequence will be amplified.
Two applications of PCR are:
- Diagnosis of infectious diseases: PCR can be used to detect the presence of specific pathogens in clinical samples, such as blood or saliva. The amplified DNA fragments can be detected using various methods, such as gel electrophoresis, fluorescent dyes, or real-time PCR.
- Forensic analysis: PCR can be used to amplify DNA samples from crime scenes or other sources, allowing for identification of individuals based on their DNA profiles. This technique is commonly used in paternity testing and criminal investigations.
Overall, PCR is a versatile and powerful technique that has revolutionized many areas of molecular biology and biomedical research.