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Polymerase Chain Reaction

PCR or Polymerase Chain Reaction is a technique used in molecular biology to generate many copies of a single piece of DNA. The method was developed in 1983 by American pharmacist Kerry Mullis. PCR made it possible to produce millions of copies of a small piece of DNA. This material is often used in molecular and biotechnology laboratories.

Polymerase Chain Reaction

Principle of PCR

The PCR technique is based on enzymatic replication of DNA. PCR uses an enzyme-mediated primer to amplify a short segment of DNA. DNA polymerase makes new strands of DNA to complete the template DNA.

The DNA polymer can only add a nucleotide to an already existing 3′-OH group. Therefore, an introduction is required. Thus, multiple nucleotides are added to the 3′ main end of the DNA polymer.

Components Of PCR

  • DNA Template – DNA of interest from the sample.
  • DNA Polymerase – Taq Polymerase is used. It is tropical and does not change at very high temperatures.
  • Oligonucleotide primers – These are small pieces of single-stranded DNA that complement the 3′ ends of the restriction strands.
  • Deoxyribonucleotide triphosphates – These provide the energy for polymerization and are the building blocks for DNA synthesis. These are each of the basics.
  • The buffer system – Magnesium and potassium provide excellent conditions for DNA modification and regeneration. It is also important for reliability, polymer activity and stability.

Types of PCR

  • In this type of fluorescence reporter, DNA amplification is detected in real time. The signal intensity of the fluorescence reporter is directly proportional to the number of amplified DNA molecules.
  • It was designed to promote communication and interaction. They reduce nonspecific binding of products due to unexpected strengthening of initial binding sites.
  • This is used to amplify multiple targets in some PCR experiments. It amplifies several different DNA sequences simultaneously.
  • It uses DNA amplification linearity to identify, characterize and quantify a sequence detected in a sample.
  • It is a PCR method for DNA fingerprinting. It uses primers whose DNA sequence is chosen randomly.

PCR Steps

  • Denaturation occurs when the reaction mixture is heated to 94°C for about 0.5 to 2 minutes. This breaks the hydrogen bonds between the two strands of DNA, making it single-stranded DNA.
  • Now the monomer acts as a template to form new strands of DNA. Heat must be supplied for a long time to ensure separation of the two wires.
  • The reaction temperature drops to 54-60 degrees C for about 20-40 seconds. Here, the primers bind to their complementary sequence on the template DNA.
  • Primers are single-stranded sequences of DNA or RNA that are 20 to 30 bases in length.
  • They act as promoters for DNA synthesis.
  • The two separated wires run in opposite directions and therefore there are two leads – forward lead and reverse lead. In this stage the temperature rises to 72-80 °C. Bases are added to the 3′ end of the primer by the enzyme Taq polymerase.
  • This makes the DNA 5′ by 3′ long. DNA polymerase increases about 1000bp/minute under optimal conditions.
  • Taq Polymerase can withstand very high temperatures. It attaches to a primer and adds DNA bases to the strand. As a result, a double-stranded DNA molecule is obtained.
  • These three steps are repeated 20-40 times to reach several DNA sequences of interest in a relatively short time.

 

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