Amplification of Gene Of Interest Using PCR | polymerase chain reaction | Manish Mevada

Amplification of Gene of Interest Using PCR

• The Polymerase Chain Reaction or PCR, as it is commonly called, was originally invented by Kary Mullis in 1985.
• Kary Mullis shared the Nobel Prize with Michael Smith in Chemistry in 1992 PCR is best defined as the DNA replication in vitro.
• It results in the selective amplification of a specific region of a DNA molecule and so can also be used to generate a DNA fragment for cloning.
• Tinc basic principle underlying this technique is that when a double - stranded DNA molecule is heated to a high temperature, the two DNA strands separate giving rise to single - stranded DNA molecules.
• If these single - stranded molecules are copied by a DNA polymerase, it would lead to duplication of the original DNA molecule and if these events are repeated many times, multiple copies of the original DNA sequence can be generated.
• The basic require ments of a PCR reaction are the following :
• DNA Template . Any source that contains one or more target DNA molecules to be amplified can be taken as template
• Two Nucleotide Primers . Primers, which are oligo - nucleotides, that hybridize to the target DNA region, one to each strand of the double helix are required.
• These primers are oriented with their ends facing each other allowing synthesis of the DNA towards one another
• Enzyme . DNA polymerase which is stable at high temperatures ( > 90 ° C ) is required to carry out the synthesis of new DNA.
• The DNA polymerase like Taq polymerase is generally used in PCR reactions which is isolated from a bacterium Thermus aquaticus.
• Other thermostable ( temperature remains stable ) polymerases can also be used

Working Mechanism of PCR

• A single PCR amplification cycle involves three basic steps, denaturation, annealing and extension (polymerisation).

Denaturation (Melting of Target DNA)

• In the denaturation step, the target DNA is heated to a high temperature (usually 94º to 96 ° C), resulting in the separation of the two strands.
• Each single strand of the target DNA then acts as a template for DNA synthesis

Annealing (Anneal = Join)

• In this step, the two oligo - nucleotide primers anneal (hybridize) to each of the single stranded template DNA since the sequence of the primers is complementary to the 3 ' ends of the template DNA.
• This step is carried out at a lower temperature (usually 40 ° to 60 ° C) depending on the length and sequence of the primers.

Extension ( Polymeri - sation)

• The final step is extension, wherein Taq DNA polymerase (of a thermophilic bacterium Thermus aquaticus) synthesizes the DNA region between the primers, using DNTPs (deoxynucleoside triphosphates) and Mg2 +.
• It means the primers are extended towards each other so that the DNA segment lying between the two primers is copied.
• The optimum temperature for this polymerization step is 72 ° C.
• To begin the second cycle, the DNA is again heated to convert all the newly synthesized DNA into single strands, each of which can now serve as a template for synthesis of more new DNA.
• Thus the extension product of one cycle can serve as a template for subsequent cycles and each cycle essentially doubles the amount of DNA from the previous cycle.

Application of PCR

• Some of the areas of application of PCR are briefly mentioned here
• Detection of Pathogens
• In recent times , PCR is being used in the detection of HIV (Virus of AIDS).
• Diagnosis of Specific Mutation
• Mutations are related to genetic diseases. By using PCR phenylketonuria, muscular dystrophy , sickle cell anaemia, hepatitis, chlamydia and tuberculosis can be diagnosed.
• PCR is also used in DNA Fingerprinting.
• Detection of Specific Microorganisms
• In recent years, PCR has also found useful for detecting specific microorganisms.
• In Prenatal Diagnosis
• It is useful to detect genetic disease in foetus before birth.
• Diagnosis of Plant Pathogens
• Many diseases of plants can be detected by using PCR
• For examples, viroids (associated with apple, grape, citrus, pear, etc.), viruses (like TMV, bean yellow mosaic virus etc), bacteria, mycoplasmas, etc.
• In Palaeontology
• PCR is used to clone the DNA fragments from the mummified remains of humans and extinct animals like wooly mammoth and dinosaurs
• Gene Therapy
• PCR proves to be of immense help in monitoring a gene in gene in gene therapy experiments.

========================================

Mail- indiabiologymanishmevada@gmail.com

Subscribe and Follow For more knowledge of Biology

Join With Me Just Click Hear

Manish Mevada

M.Sc, M.Phil, B.Ed

THANK YOU.