Real-time polymerase chain reaction

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In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is a laboratory technique used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction.

The procedure follows the general pattern of polymerase chain reaction, but the DNA is quantified after each round of amplification; this is the "real-time" aspect of it. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.

Frequently, real-time polymerase chain reaction is combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type.

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 known as RT-PCR.

Contents 1 Background 2 Real-time PCR using Double-stranded DNA Dyes 3 Fluorescent reporter probe method 4 Applications of real-time polymerase chain reaction 5 References 6 Further reading 7 External link

[edit] Background

Cells in all living organisms regulate their cellular activities by activating or deactivating the expression of their genes. Gene expression corresponds to the number of copies of messenger RNA (mRNA) that exist for a particular gene. As mRNA becomes transcribed at the ribosome to produce functional proteins, mRNA levels tend to roughly correlate with protein expression.

Traditionally, the amount of a particular mRNA produced, and thus the activation status of a gene has been measured by a technique known as northern blotting. In this method, purified RNA is separated by agarose gel electrophoresis, and then probed with a specific anti-sense DNA probe for the gene of interest. Although this technique is still used to measure gene expression, it requires relatively large amounts of RNA and thus cannot be performed when tissue samples are limited.

In order to detect gene expression at minute levels from single or small numbers of cells, some amplification is necessary. The polymerase chain reaction is an effective tool for amplifying DNA, however for this to be adapted to measure RNA, the RNA sample first needs to be reverse transcribed to DNA via an enzyme known as a reverse transcriptase. This transcribed DNA is known as cDNA or complementary DNA. This method, known as RT-PCR, required extensive optimisation of the number PCR cycles, so as to obtain results during logarithmic DNA amplification.

Development of PCR technology that uses fluorophores to measure DNA amplification in real-time allows researchers to bypass the extensive optimisation associated with normal RT-PCR. In real-time PCR, the amplified product is measured at the end of each cycle. This data can be analysed by computer software to calculate relative gene expression between several samples, or mRNA copy number based on a standard curve.

[edit] Real-time PCR using Double-stranded DNA Dyes

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, dsDNA dyes such as 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 in a thermocycler, and after each cycle, the levels of fluorescence are measured with a camera; the dye only fluoresces when bound to the dsDNA ( i.e. PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.

Like other real-time methods, the quantity you initially get does not have an absolute unit associated with it (i.e. mRNA copies/cell). As described above, a comparison of your sample to a standard dilution will only tell you what fraction or ratio your sample has in comparison to a 1X, 0.1X, 0.01X, 0.001X, etc. diluted sample. For example, you can say liver tissue expresses a specific enzyme twice as much as lung tissue. But there's a caveat to this, depending on how much tissue you started with your calculation can be way off.

The best way to ensure accuracy is to normalize your sample with a stably expressed gene often called a housekeeping gene. It is assumed the housekeeping gene does not vary in quantity and thus can be used to precisely measure total amount of sample. In your calculations you divide your measurement by the housekeeping gene to account for variations in sample amount. For example, if you use ~0.10 ng of tissue in one sample and ~0.11 ng of tissue in another, your housekeeping gene will correct this problem.

[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.

[edit] Applications of real-time polymerase chain reaction

There are numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and research applications.

Diagnostically real-time PCR is applied to rapidly detect the presence of genes involved in infectious diseases, cancer and genetic abnormalities. In the research setting, real-time PCR is mainly used to provide highly sensitive quantitative measurements of gene transcription.

The technology may be used in determining how the genetic expression of a particular gene changes over time, such as in the response of tissue and cell cultures to an administeration of a pharmacological agent, progression of cell differentiation, or in response to changes in environmental conditions.

Also, the technique is used in Environmental Microbiology, for example to quantify resistence genes in water samples.

[edit] References

• Molecular Biology of the Cell, Alberts, B. et. al, Garland Science
• A-Z of Quantitative PCR, Bustin, S.A. (ed.), IUL Biotechnology, 2004
• The Real-Time Polymerase Chain Reaction, Kubista, M. et. al, Molecular Aspects of Medicine 27, 95-125 (2006)

[edit] Further reading

• Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. (1992). "Simultaneous amplification and detection of specific DNA sequences." Biotechnology 10:413–417.
• Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). "Kinetic PCR: Real time monitoring of DNA amplification reactions." Biotechnology 11:1026–1030.
• Wawrik B, Paul JH, Tabita FR (2002) Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68:3771-3779.

[edit] External link

• Real Time PCR Tutorial by Dr Margaret Hunt, University of South Carolina, September 5, 2006
• Real-time PCR Animation - PCR and Real-time PCR principles and comparison