Genetics

Real Time Polymerase Chain Reaction Pcr



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Real-time PCR is a relatively new molecular biology technique that quantifies the amount of a particular piece of DNA in a sample. This article will describe how this process works, and will look at what applications this technology has found.

Regular "vanilla" PCR amplifies a sequence of DNA between two complementary synthetic DNA primers. Denaturation of the starting DNA rips apart the double stranded DNA helix using heat. Then the primers can bind to their sequences at each end, and a polymerase (taq) fills in the bits between the primers. Thermal cycling doubles the amount of DNA each cycle - meaning you can make LOTS of your DNA quite easily and quickly.

Real-time PCR works similarly, but the amplification can be tracked in real-time. Two techniques are used to facilitate this. Firstly, a compound (often SYBR green) can be added to the PCR mix. This fluoresces when excited by light ONLY when it is bound to double stranded DNA. Hence, the degree of fluorescence is proportional to the amount of dsDNA generated.

The other technique uses a probe with a sequence complementary to a region in the middle of your sequence. This probe has 2 molecules attached to it at opposite ends. At one end, there is a chromophore which fluoresces when excited, and at the other end of the probe there is a quencher which stops the chromophore fluorescing. These two molecules are very close together when attached to the probe so the quencher can do it's job really easily. However, when the taq fills in the sequence between the two primers, it destroys the probe, so the quencher is no longer attached to the chromophore. This allows the chromophore to fluoresce and this is read by the real-time PCR machine.

Both these technique generate a curve which starts off at low fluorescence and increases before leveling off as reagents become limiting. The time until 1/2 maximum fluorescence gives an indication of how much DNA you had to start with. If it takes a long time, you didn't have much DNA to start with. By running standards at the same time as your samples, you can quantify how much DNA you had.

Real-time PCR is often used to quantify gene expression. mRNA is reverse transcribed to cDNA, then this is real-time PCRed. Controls are very important here as the efficiency of the reverse transcription step can vary greatly.

Another use of real-time PCR is in ancient DNA studies. If an ancient DNA study that is trying to obtain sequences from old bones, for example, started from only a VERY low number (ie. less than 100), the sequence may have many mistakes. By quantifying the amount of starting material, this problem can be picked up, and the sequence can be treated with the appropriate skepticism.

That's a whorl-wind tour through real-time PCR - if you have any questions you can contact me and I'll be happy to answer them.

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