Help with Real Time-PCR (RT-PCR)

A previous lecture on diagnosing bacteria referred to "RT-PCR", and this was described as "Reverse Transcriptase - PCR", which is correct.  Reverse Transcriptase - PCR is used to detect mRNA to monitor gene expression.  The mRNA is first converted to cDNA by treating it with Reverse Transcriptase, an enzyme from viruses that copies RNA to DNA.  After the RNA to DNA conversion, the cDNA is then amplified by the conventional PCR procedure, of which you should already be familiar and which is described in the text (pages 179-180).

Unfortunately, Real Time - PCR is also abbreviated "RT-PCR", so there is understandable confusion with his term.  However, Reverse Transcriptase - PCR and Real Time - PCR are very different.  Since Real Time - PCR is likely to become a common diagnostic tool during soon, you should be familiar with the basics. 

In traditional PCR, specific DNA sequences are amplified using repeated rounds (usually 25 to 30) of denaturation, primer annealing, and extension by thermostable DNA polymerase to generate a specific fragment of DNA which is the examined by gel electrophoresis.  This can take many hours and is labor intensive.

In contrast, with Real Time - PCR special methods are used to detect and quantify the generation of the amplified DNA product without having to wait until the full set of amplification rounds is completed and without having to run a gel, hence the name "Real Time".  The generation of amplified DNA is monitored by light emitted directly from the reaction tube using sensitive optics and computers.  A curve is generated for each reaction, and the speed at which detectable product is generated gives an indication of the amount of template DNA (target) was present in the initial sample.  Hence, Real Time - PCR is both qualitative (was the target DNA there or not?) and quantitative (if it was there, how much was there?).  Because of the speed and sensitivity of Real Time - PCR, you will likely experience diagnosis of infectious diseases by this procedure in your practices.

Perhaps the best method for detecting product is the TaqMan system sold by Applied Biosystems.  It is described in this simple animation at the bottom of this linked page from Johns Hopkins University.  Another method from Applied Biosystems uses a fluorescent dye (SYBR Green) that labels double-stranded DNA as it is produced during the amplifications (much cheaper but more prone to errors).  A third method is called "molecular beacons".