Definition of PCR Requirements for PCR PCR Process Agarose gel electrophoresis.

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Research Techniques Made Simple: Polymerase Chain Reaction

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Presentation transcript:

Definition of PCR Requirements for PCR PCR Process Agarose gel electrophoresis

The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It is used to exponentially amplify a fragment of DNA by in vitro enzymatic replication. PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as

the sequencing of genes and the diagnosis of hereditary diseases,sequencing hereditary diseases the identification of genetic fingerprints.genetic fingerprints the detection and diagnosis of infectious diseases.infectious diseases and the creation of transgenic organisms.transgenic organisms

DNA template that contains the DNA region to be amplified. One or more primers, which are complementary to the DNA regions at the 5' (five prime) and 3' (three prime) ends of the DNA region.primers5'3' a DNA polymerase such as Taq- polymearaseDNA polymerase (dNTPs). Buffer solution thermal cycler

1) Denaturation: During denaturation, the template DNA is separated (denatured) into its two separate strands by heating at the temperature about 95º C. 2) Annealing: This involves the annealing of the primer to the denatured DNA. The temperature is lowered to a degree specific for the primer, which generally lies between 40ºC and 68ºC. 3) Extension: The third step, the synthesizing, takes place at a temperature of around 72º C. This corresponds to the optimal temperature for the Taq- polymerase to work.

For visualization of DNA, ethidium bromide is added to DNA to gives a fluorescence, which can be seen under a UV light. Nucleic acids have consistent negative charge imparted by their phosphate backbone, and migrate toward the anode Small fragments of DNA can move more easily through the gel than larger ones and so all the different sizes spread out and separation occurs on the basis of size.

The result