Thursday, January 16, 2014

Real Time PCR (qPCR)

I don't know why, but, I really love this guy! :P Yes, he is PCR! You ask me anything, like, what technique we can use for introducing mutation? what for diagnosis? I'll answer PCR for all! :P Let me talk about him for sometime here.

PCR (Polymerase Chain Reaction) is used for amplifying any gene from a given sample using Taq polymerase, dNTPs, Primers and buffers. After PCR the amplified product or amplification is generally checked by running an Agarose gel electrophoresis. But, in case of Real Time PCR there is no need for running gels as the progress of the PCR is monitored online with the help of fluorophores. Generally SYBR Green dye, Taqman or molecular beacon probes are used for Real Time PCR. In SYBR Green method, the fluorophore binds with the double stranded DNA and produces fluorescence, and hence as the amplification increases, fluorescence increases, but the fluorophore has no specificity and hence, even if the amplified product is not the product of your interest you would find increase in fluorescence. The TaqMan and molecular beacons are probes which bind which are labeled with fluorescent dyes at one end and quencher at other end. When the fluorescent dye and the quencher are at close proximity there won’t be any fluorescence but on amplification the fluorescent dye and the quencher gets apart thus causing increase in fluorescence. These are specific to the gene amplified and hence we could monitor the amplification specifically online.

Another method is LUX (Light upon eXtension) in which the primer is labeled with fluorophore at the 3’end. The primer remains in a hair pin like structure initially, and, at that configuration it emits less fluorescence. When the primer becomes linear single strand, the fluorescence increases and when it gets extended and becomes double strand, the fluorescence increases further. As the primers are specific, the fluorescence increases only when the amplification is specific.


Melting curve analysis is generally done for the PCR products in case of LUX and SYBR Green as the fluorescence could also be due to the primer dimer. In the melting curve analysis, the product is heated thus allowing it to unwind, the melting point temperature (Tm time taken for half of the sample to unwind as single strand) is noted. The melting temperature will be different for primer dimers and the product as they differ in length. And hence, fluorescence formed could be exactly correlated only with the product after this analysis. Many of the light cyclers have inbuilt option for performing this melting curve analysis.

It has various applications; few of them are listed below:

  • Diagnosis where genes which are specific for a particular species can be targeted and amplified, if the amplification is positive then the sample could be diagnosed as infected. The specialty of qPCR is that it also gives exact severity of infection.
  • Microbiology – for comparing the differences between different species, sub species and serovars of microbes
  • Research  - for works like cloning amplification of a particular gene is essential and also for measuring expression of a particular gene qPCR can be used.
Hope, you also loved him! ;) 

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