PCR

• Polymerase Chain Reaction.
• PCR is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. 
• Developed in 1983 by Kary Mullis
• A basic PCR set up requires several components and reagents. These components include
  • DNA template that contains the DNA region (target) to amplify
  • Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Primers can be custom made in a laboratory that are complementary to the DNA segment to be amplified.
  • Taq polymerase (^[1] as the DNA Polymerase can not attach to a DNA strand and elongate on its own. It should also be heat resistant, so that it can withstand the denaturation process.
  • Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
  • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
  • Bivalent cations, magnesium or manganese ions; generally Mg²⁺ is used, but Mn²⁺ can be used for PCR-mediated DNA mutagenesis, as higher Mn²⁺ concentration increases the error rate during DNA synthesis
  • Monovalent cation potassium ions
• Procedure
  • Initialization step (Only required for DNA polymerases that require heat activation by hot-start PCR.): This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes.
  • Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
  • Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should only bind to a perfectly complementary part of the template. If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind. Typically the annealing temperature is about 3–5 °C below the Tm of the primers used. Stable DNA–DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation. It is very vital to determine the annealing temperature in PCR. This is because in PCR, efficiency and specificity are affected by the annealing temperature. An incorrect annealing temperature will cause an error in the test.
  • Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to amplify. As a rule-of-thumb, at its optimum temperature, the DNA polymerase polymerizes a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
• Applications
  • Selective DNA isolation : PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization robes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region.
  • Amplification and quantification of DNA : PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. And quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample
  • Medical application

 

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