Polymerase Chain Reaction (PCR) is a commonly used laboratory technique for the diagnosis of a wide variety of genetic testing and infectious disease. The PCR can detect the DNA of microbial pathogens such as bacteria and viruses in clinical specimens with high sensitivity. PCR is a technique where specific fragments of DNA can be amplified which validate the presences of DNA or RNA of pathogens in a patient’s specimen.
A basic PCR set-up requires several components and reagents which includes DNA template that contain the target region to amplify, DNA polymerase, primers, dNTPs and buffer solution to provide suitable chemical environment for optimal activity and stability of DNA.
Typically, the PCR consists of a series of temperature changes called thermal cycles with each cycle consisting of 2 or 3 discrete temperature steps. The different steps involve are as follows.
Denaturation is the first step which causes DNA melting or denaturation of double stranded DNA by breaking of the Hydrogen bonds between bases.
Annealing is the next step where the temperature is lowered allowing annealing of the primers to each of single stranded DNA template.
The final step is Extension or Elongation which takes place at an optimal temperature (72 degrees). In this step the DNA polymerase synthesis new DNA strand by adding dNTPs to the complementary DNA templates.
With each successive cycle the newly created DNA will become the template for the next cycle leading to an exponential amplification of the specific target DNA region. The cycle repeats for 25-35 times in a typical reaction and would take 2-4 hours depending on the length DNA region being copied. At the end of the reaction the target region can go from one to few billion copies.