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| Project C 4 |
| Part 1: Principle of real time quantitative PCR |
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a) The TaqMan method using the conventional primer pair in combination with a third oligonucleotide, which is 5´-labelled with a fluorescence dye (FAM) and 3´-labelled with a fluorescence quencher (TAMRA). During the extension step the probe is degraded due to the 5´-3´-exonuclease activity of the Taq-DNA-polymerase leading to a spatial separation of dye and quencher and therefore to an increase in fluorescence (Figure 1).
b) The SYBR-Green method uses SYBR-Green I® as a fluorescence dye which intercalates specifically with dsDNA during the extension phase of the PCR.
For quantification of the starting amount of template DNA the threshold cycle (Ct) for each sample is calculated. The Ct-value is the cycle number when the fluorescence of FAM or SYBR-Green measured is significantly different to the background level for the first time. Ct-value is proportional to the logarithm of initial template DNA concentration.
Figure 1:
principle of the TaqMan assay using a fluorogenic probe labeled with the fluorescence dye FAM (F) and a fluorescence quencher TAMRA (Q).
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