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qPCR / Real Time Quantitative PCR

Real Time PCR, also known as quantitative PCR or qPCR, is an effective tool to make the in-vitro amplification of specific DNA fragments visible without using an agarose gel. It can also be described as the simultaneous amplification and detection / quantification of nucleic acids by means of the polymerase chain reaction (PCR).

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By using Real Time PCR instruments amplification and detection can be combined in a single step. An additional, time-consuming and not easily quantifiable gel electrophoresis is not required.

In Real Time Quantitative PCR different fluorescent dyes and approaches are used to trace the progress of amplification during the Real Time Quantitative PCR reaction and to get a strong increase in fluorescence during amplification.

To be able to measure an increase in fluorescence there must be a mechanism that guarantees almost no fluorescence at the beginning of the amplification (no amplified DNA) but must also lead to a measurable increase if specific DNA is amplified.

Today 2 principle approaches are used to measure the amplification progress in Real Time Quantitative PCR in-vitro:

1. Use of fluorescent labeled probes in Real Timne Quantitative PCR
- Real Time Quantitative PCR with TaqMan® probes, consist of a fluorophore at one end of the primer and a quencher at the other end of the same primer in a distance that enables the quencher to quench the fluorescence signal. During the Real Time Quantitative PCR the 3’-5’ exonuclease acitivity of the polymerase degrades these primers thus releasing the fluorescent dye and quencher to the surrounding PCR reaction solution. After getting released the quencher will move away from the fluorescent dye molecule thus loosing its ability to quench fluorescence. The more qPCR proceeds the more primer has been degraded and the more free fluorescent dye is in solution, which increases total fluorescence drastically.

- Real Time Quantitative PCR with Molecular Beacons (probes with fluorescent dyes and quencher forming hairpin structures) consist of a fluorophore at one end of the primer and a quencher at the other end of the same primer. The primary sequence of the primer allows to "self-prime" at low temperatures, forming a hairpin structure which brings the fluorescent dye molecule and the quencher in direct neighbourhood thus enabling the quencher to quench the fluorescence signal. The hairpin structure will be destroyed during elevated temperature (denaturation) resulting in a primer that can bind now to single DNA strands instead of priming with itself. Upon cooling, the probe binds to the complementary DNA much faster as the complementary stretch is about 18 bases instead only 5-8 base pairs for the hairpin structure. If the Molecular Beacon binds to DNA the fluorescent dye is no longer suppressed by the quencher molecule and thus a fluorescence signal can be detected by the PCR instrument. The more amplified DNA is available the more Molecular Beacon probes will bind to DNA and the more the fluorescence signal will increase.

Besides the above described fluorescence probes Real Time Quantitative PCR needs special DNA-amplifying enzyme, which are supported as ready-to-use qPCR master mix. qPCR master mixes are pre-mixed PCR mixes which contain all necessary components for Real Time Quantitative PCR, with the exception of primer and DNA template to proceed with the qPCR. qPCR Mastermixe are offered commercially from different companies. For use in qPCR Genaxxon offers the following qPCR master mixes. M3010 (High ROX)M3031 (Low ROX) - M3045 (No ROX). As master mixes have to be stored cooled Genaxxon has developed a lyophilized (freeze- dried) qPCR master mix which is stable at ambient temperature for at least 24 months.

2. Use of non-specific intercalating fluorescent dye in Real Time Quantitative PCR
The above described methods are very sensitive and specific but also relative expensive as DNA-probes are no cheap products. To minimize the cost issue a non-specific intercalating fluorescent dye in Real Time Quantitative PCR with is used. This works as the fluorescence of the different available intercalating dyes is very low without binding to DNA but will increase dramatically if these dyes are bound to DNA. By binding to DNA and subsequent conformational change, a significant increase in fluorescence can be measured, whereby the measured fluorescence can be correlated with the amount of DNA built.

Appropriate qPCR master mixes which already contain the intercalating fluorescence dye are offered commercially. Genaxxon for example offers the following products: M3011 (Low ROX) - M0323 (No ROX) - M3052 (High ROX).

Real Time Quantitative PCR instruments measure the increase in fluorescence during the PCR reaction and will do the rapid quantification by mathematical algorithms that allow the accurate determination of Ct-values or will measure the melting temperature of double stranded DNA.

By appropriate calibration of the PCR instruments this offers the possibility to calculate how much DNA (or RNA) the original sample contained/contains.

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