What is PCR?
PCR (Polymerase Chain Reaction) is a laboratory technique widely used in microbiology and other fields of molecular biology. It is a method for amplifying specific DNA sequences, allowing researchers to produce large amounts of DNA from a small initial sample. PCR is highly sensitive and can detect and amplify even trace amounts of DNA.
The PCR process involves a series of temperature cycles that enable DNA replication. It requires a DNA template, primers (short DNA sequences that bind to the target DNA region), DNA polymerase (an enzyme that synthesizes new DNA strands), and nucleotides (building blocks of DNA).
Here's a breakdown of the typical temperature cycles in PCR:
Denaturation: This step involves heating the PCR reaction mixture to a high temperature, usually around 94-98 degrees Celsius. The purpose of denaturation is to separate the double-stranded DNA into single strands by breaking the hydrogen bonds between the nucleotide bases. This step ensures that the DNA template is available for the next steps.
Annealing: After denaturation, the reaction temperature is lowered to a specific range, typically around 50-65 degrees Celsius. During annealing, short DNA primers specifically designed to bind to the target DNA sequence attach to their complementary sequences on the single-stranded DNA template. The primers provide the starting point for DNA synthesis by DNA polymerase.
Extension: The temperature is raised to the optimal activity temperature of the DNA polymerase used in the PCR, usually around 72 degrees Celsius. The DNA polymerase synthesizes new DNA strands using the single-stranded DNA template and the bound primers. The polymerase adds nucleotides to the growing DNA chain in a 5' to 3' direction, extending the primers and copying the target DNA sequence.
By repeating these temperature cycles, each cycle doubles the amount of DNA present, resulting in exponential amplification of the targeted DNA sequence. The number of cycles performed is determined by the specific application and desired level of amplification.