Quantitative polymerase chain reaction

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Quantitative polymerase chain reaction (Q-PCR) is a modification of polymerase chain reaction used to rapidly measure the quantity of a product of polymerase chain reaction. It is preferably done in real-time, thus is an indirect method for quantitatively measuring starting amounts of DNA, complementary DNA or ribonucleic acid (RNA). It is commonly used to determine whether a genetic sequence is present, and if it is present, to determine the number of copies in the sample. There are 3 methods which vary in difficulty and detail. Like other forms of polymerase chain reaction, the process is used to amplify DNA samples, via the temperature-mediated enzyme DNA polymerase.

The three commonly used methods of quantitative polymerase chain reaction are through agarose gel electrophoresis, the use of SYBR Green, a double stranded DNA dye, and the fluorescent reporter probe. The latter two of these three can be analysed in real-time, forming real-time polymerase chain reaction.

Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not to be confused with reverse transcription polymerase chain reaction, also referred to as RT-PCR.

[edit] Agarose gel electrophoresis method

Agarose gel electrophoresis is the simplest method, but also the slowest and least accurate, depending on the running of an agarose gel via electrophoresis. It cannot give results in real time.

1. The unknown sample and a known sample are prepared with a known concentration of a similarly sized section of target DNA for amplification.
2. Both reactions are run for the same length of time in identical conditions (preferably using the same primers, or at least primers of similar annealing temperatures).
3. Using agarose gel electrophoresis separate the products of the reaction from their original DNA and spare primers.
4. The relative quantities of the known and unknown samples are measured to determine the quantity of the unknown.

This method is generally used as a simple measure of whether the probe target sequences were present or not, and rarely as 'true' Q-PCR. It has been superseded by the other, real-time, methods. It is, at best, a semi-quantitative method.

[edit] SYBR Green method

Using SYBR Green dye is more accurate than the gel method, and gives results in real time. A DNA binding dye binds all newly synthesized double stranded (ds)DNA and an increase in fluorescence intensity is measured, thus allowing initial concentrations to be determined. However, SYBR Green will label all dsDNA including any unexpected PCR products, leading to potential complications.

1. The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
2. The reaction is run, and the levels of fluorescence are monitored; the dye only fluoresces when bound to the dsDNA. With reference to a standard sample, the dsDNA concentration in the PCR can be determined.

[edit] Fluorescent reporter probe method

Using fluorescent reporter probes is the most accurate and most reliable of the methods, but also the most expensive. It uses a sequence specific RNA or DNA based probe so as to only quantify the probe sequence and not all double stranded DNA.

It is commonly carried out with an RNA based probe with a fluorescent reporter and a quencher held in adjacent positions. The close proximity of the reporter to the quencher prevents its fluorescence, it is only on the breakdown of the probe that the fluorescence is seen. This process depends on the 5' to 3' exonuclease activity of the polymerase involved.

1. The reaction is prepared as usual, with the addition of the probe.
2. The reaction commences. On melting of the DNA the probe is able to bind to its complementary sequence in the region of interest of the template DNA (as the primers will too).
3. When the PCR is heated to activate the polymerase, the polymerase starts writing the complementary strand to the primed single strand DNA. As the polymerisation continues it reaches the probe bound to its complementary sequence. Chemically like an existing RNA primer the polymerase "overwrites" the probe, breaking it into separate nucleotides, and so separating the fluorescent reporter and the quencher. This results in an increase in fluorescence.
4. As PCR progresses more and more of the fluorescent reporter is liberated from its quencher, resulting in a well defined geometric increase in fluorescence. This allows accurate determination of the final, and so initial, quantities of DNA.

Polymerase chain reaction techniques
Quantitative polymerase chain reaction (Q-PCR) | Real-time polymerase chain reaction (QRT-PCR) | Reverse transcription polymerase chain reaction (RT-PCR) | Inverse polymerase chain reaction | Nested polymerase chain reaction | Touchdown polymerase chain reaction