Polymerase Chain Reaction


The polymerase chain reaction is a technique for cloning a particular piece or amplifying a single or few copies of a piece of DNA, generating millions or more copies of that particular DNA sequence. One can make virtually unlimited copies of a single DNA molecule even though it may initially be present in a mixture containing many different DNA molecules.

In PCR, the enzyme DNA polymerase assembles a new DNA strand from nucleotides, by using single-stranded DNA as a template and DNA primers for initiation of DNA synthesis. Developed in 1984 by Kary Mullis, PCR is now a common technique used in biological research.

MATERIAL REQUIRED

A basic PCR requires the following components:

  • DNAtemplate that contains the DNA region called target to be amplified.
  • Two primers complementary to the DNA regions at the 5′ or 3′ end of DNA.
  • The enzyme, Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
  • Deoxynucleoside triphosphates (dNTPs) that are building blocks for DNA synthesis, e.g. dATP, dGTP, dCTP and dTTP.
  • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
  • Divalent cations, magnesium or manganese ions; generally Mg2+ is used but Mn2+ can be utilized for PCR-mediated DNA mutagenesis.
  • Monovalent cation potassium ions.

THE PROCEDURE

In order to perform PCR, you must know at least a portion of the sequence of the DNA molecule that you wish to replicate. You must then synthesize primers that are short oligonucleotides, containing about two dozen nucleotides that are precisely complementary to the sequence at the 3′ end of each strand of the DNA you wish to amplify.

The DNA sample is heated to separate its strands and mixed with the primers.

If the primers find their complementary sequences in the DNA, they bind to them.

Synthesis begins in 5′ – 3′ direction, using the original strand as template.

The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) and a DNA polymerase that is not denatured by the high temperature. Almost all PCRs employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles.

The first step consists of heating the reaction to a temperature of 94-98°C, which is held for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

In the second 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. 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 synthesis.

In the third step, the temperature is raised commonly to 72°C and for Taq polymerase to 75-80°C. DNA polymerase now 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 strand

Fourth step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Polymerization continues until each newly-synthesized strand has proceeded far enough to contain the site recognized by the other primer.

In the final step temperature is lowered at 4-15°C for an indefinite period for short-term storage of the reaction.

Now you have two DNA molecules identical to the original molecule.

You take these two molecules, heat them to separate their strands, and repeat the process. Each cycle doubles the number of DNA molecules. Using automated equipment, each cycle of replication can be completed in less than 5 minutes. After 30 cycles, a single molecule of DNA has been amplified to more than a billion copies.




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