Real Time PCR Probes

Hi there, i have talked about real time PCR in my previous post. In order to get results from RT-PCR, probes such as Taq man are used.

Taq Man is a RT Probe (the Cheaper one). Taq man is a DNA sequence with binded 2 proteins Reporter protein (at the 5' end) and quencher protein at the 3' end. The quencher protein will inhibit the reporter protein when they are both in the same DNA strand. This is due to the inhibition of proton transfer.

Basic: PCR processes are Denaturation, Annealing and Amplification.
At denaturation, DNA will become Single stranded DNA strands. At Annealing, the probes will bind to complementary base pairs along the targetted DNA strand. At Amplification, Taq Polymerase will bind to the DNA strands and produce copies of the DNA strands. The Taq polymerase will move down the targetted DNA strands to produce complementary strands. Once the Taq Polymerase will "free" the reporter protein once it hits the probe (Breaking up the probe). The released Reporter protein will fluores to give a signal. A reader in the machine will pick up the signal and the signal will be converted to picture form which is shown on the computer screen. So as the PCR cycles continues more reporter proteins will be released thus forming a graph.

Another kind of probe which is used is the molecular beacon (slightly more expensive i heard). Molecular beacons are in a hair pin structure with the 2 proteins at the ends of the DNA seqence. It will denature at the Denaturation step to form SSdna. Will bind to DNA at annealing and the reporter protein will be activated to give off the fluorescence signal.

The two probes above are the more commonly used probes due to the cost.
For pictorial form, please refer to the link below
http://www.iba-biotagnology.com/images/naps/rt_pcr.gif (Taq Man)
http://www.pentabase.com/Portals/0/EasyBeacons%20mekanisme.jpg (Molecular beacon)

Credits to
Dr. Chan for teaching me about RT-PCR on first week of SIP.

Cheers
Tiong Han
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