REAL TIME PCR and its Application notes

REAL TIME PCR 

  • Real-time polymerase chain reaction (qPCR) is commonly used to measure gene expression. It is more sensitive than micro arrays in detecting small changes in expression but requires more input RNA and is less adaptable to high-throughput studies.
  •  It is best suited for studies of small subsets of genes. 
  • Its one major shortcoming is that the sequence of the specific target gene of interest must be known (so you can design the PCR primers),
  •  hence real-time PCR can only be used for studying known genes. 
  • It is also known as a quantitative polymerase chain reaction (qPCR), which is a laboratory technique of molecular biology based on the polymerase chain reaction .
PRINCIPLE OF REAL TIME PCR(qPCR).

  • This same principle of amplification of PCR is employed in real-time PCR. But instead of looking at bands on a gel at the end of the reaction, the process is monitored in “real-time”. The reaction is placed into a real-time PCR machine that watches the reaction occur with a camera or detector.

    Although many different techniques are used to monitor the progress of a PCR reaction, all have one thing in common. They all link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle. Hence, as the number of gene copies increases during the reaction, so does the fluorescence, indicating the progress of the reaction.

APPLICATION OF qPCR
  • Gene expression analysis.
    • Cancer research
    • Drug research
  • Disease diagnosis and management.
    • Viral quantification
  • Food testing.
    • GMO food
  • Animal and plant breeding.
    • Gene copy number

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