TaqMan

Taqman is a proprietary name for a means of measuring the amount of product produced, in the polymerase chain reaction.

Polymerase chain reaction

PCR is a fundamental technique in molecular biology, which (a) locates in the entire genome, the bit of DNA you want to study, ad (b) very quickly makes trillions of copies of the molecule, which gives you enough to do other experiments.

The problem with PCR is that it originaly was difficult to see if the reaction was going ahead once you had set it up. You had to wait and afterwards analyse the products.

Around 1993, several methods came on stream, using various ingenious methods with fluorescent dyes, to show whether the reaction was going ahead and what it was making. This was done by carrying out PCR in transparent plastic tubes, with a fluorimeter built into the thermal cycler, to monitor fluorescence during the reation.

Taqman is one of these. Usually things are so arranged, that as the PCR is going on, the machine includes a light, and estimates the amount of fluorescence the reaction gives off. in simple terms, if the reaction is proceeding correctly - finding the right bit of DNA and copying it - fluorescence increases, showing this.

Principle

In brief the principle is to make a probe (a synthetic bit of DNA, 20-30 bases long), with two types of dye molecule, one at each end. At the 5' end there is a standard fluorescer, such as fluorescein: if you shine say blue light on fluorescein, it fluoresces with green . At the 3' end there is another dye, called a "quencher" which soaks up the green light that fluorescein produces and itself emits at a lower wavelength, say red. Because the fluorescein and the quencher are on the same molecule of DNA, the quencher soaks up all the light that fluorescein emits. So if you shine blue light on the probe, and look for green light, you would see nothing. If you looked for red light, you might see something from the quencher.

The probe binds to the middle of the DNA sequence you are amplifying.

The probe is added at the start of reaction, along with all other ingredients, and it binds to the target DNA sequence you are trying to amplify.

During amplification, Taq polymerase -the protein which copies DNA - works its way along a DNA strand, copying it. When it finds another piece of DNA stuck there, in its way - in this case the probe, it will start to chew it up to remove it. The first thing it does, is chew up the end of the molecule with the fluorescein, and the fluorescein floats off into the solution. Now if you shine blue light on the solution, and look for green, you can see green. The fluorescein drifts away from the quencher, and eventually is so far away, that the quencher can no longer capture its light.

So to return to the big picture - this means that if you set a PCR up, you should see green fluorescence increasing as the reaction goes on, more and more PCR product is produced, more andmore probe binds to it, and more and more of the probe gets chewed up, and more fluorescein released.

origin of the name

the name follows "pacman", an early video game in which monsters found their way round a maze, chomping up things as they went.

uses of Taqman probes

The probes have two main uses

quantitative PCR

In this experiment, the experimenter sets a 'threshold' level of fluorescence, and times how many cycles of PCR it takes, for fluorescence to reach that level. From the number of cyclies it takes, the experimenter can work out how many molecules of the target DNA he started off with. This can be used for e.g measuring numbers of viruses in a sample.

identification of mutations

taqman probes are very specific for their target sequence - if the sequence is altered, the probe will not bind, and no fluorescence is seen. A typical approach to detect mutations, might be to have two Taqman probes, one that will bind to te normal sequence, and another to bind to the mutated sequence. You set up two PCR reactions, and do one with each probe. Depending on which sequence is present - normal or mutated - either the mutant probe will bind or the normal one will - and the one that binds, emits fluorescence. this can be used to detect mutations or sequence changes in DNA.

In molecular biology, quantitative real time PCR methods using a dual-labelled fluorogenic probe called a TaqMan probe is a rapid fluorophore-based real-time PCR method . The TaqMan Real-time PCR measures accumulation of a product via the fluorophore during the exponential stages of the PCR, rather than at the end point as in conventional PCR. The exponential increase of the product is used to determine the threshold cycle, C_T, i.e. the number of PCR cycles at which a significant exponential increase in fluorescence is detected, and which is directly correlated with the number of copies of DNA template present in the reaction. The set up of the reaction is very similar to a conventional PCR, but is carried out in a real-time thermal cycler that allows measurement of fluorescent molecules in the PCR tubes. Different from regular PCR, in TaqMan real-time PCR a probe is added to the reaction, i.e., a single-stranded oligonucleotide complementary to a segment of 20-60 nucleotides within the DNA template and located between the two primers. A fluorescent reporter or fluorophore (e.g., 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescin, acronym: TET) and quencher (e.g., tetramethylrhodamine, acronym: TAMRA, of dihydrocyclopyrroloindole tripeptide ‘’minor groove binder’’, acronym: MGB) are covalently attached to the 5' and 3' ends of the probe , respectively. The close proximity between fluorophore and quencher attached to the probe inhibits fluorescence from the fluorophore. During PCR, as DNA synthesis commences, the 5' to 3' exonuclease activity of the Taq polymerase degrades that proportion of the probe that has annealed to the template (hence its name: Taq polymerase + PacMan). Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Real Time Thermal Cycler

A Taqman RT-PCR thermal cycler from Applied Biosystems.

A real-time PCR cycler has a lid with built-in fiber optic cables that measure the fluorescence in the reaction tubes (using laser beams for excitation and detection of the fluorescent emission from the fluorophore). Fluorescence intensities are logged and data stored at each PCR cycle, and then used to create amplification plots of ΔRn (fluorescent signal detected - background) vs cycle number to identify the threshold cycle, C_T, which is used to quantitatively determine the amount of DNA template present in the PCR.