2. Oligonucleotides – Primers and Probes by … as quality counts! Competence and Service in Molecular Biology metabion´s history

3. Real Time/Quantitative PCR Energy shift from an electronically excited molecule (the donor fluorophore) to a neighboring molecule (the acceptor or quencher) Donor molecule returns to its ground state without emission of light (i.e., fluorescence emission). Fluorescence Resonance Energy Transfer - FRET - Principle of FRET FRET can occur when donor and acceptor molecules are in close proximity but do not require actual physical contact. In the process of FRET, de-excitation of the donor molecule is linked to excitation of the acceptor molecule. In the figure, FRET is represented by the de-excitation pathway leading from the S 1 level of the donor to the S 1 level of the acceptor. Photons of light are not involved. Once excited, the acceptor can undergo de-excitation by the same emissive and non-emissive processes described for the donor. Competence and Service in Molecular Biology

4. 1) Energy lost by de-excitation of the donor molecule, S 1 -S 0, be matched by the energy required for excitation of the acceptor  the absorption spectrum of the acceptor molecule must overlap the emission spectrum of the donor moleculethe Primary Conditions for FRET Fluorescence Resonance Energy Transfer - FRET - 3) Donor and acceptor transition dipole orientations must be approximately parallel. 4) Donor/Acceptor Pairs: In general, donor and acceptor are different dyes, each having unique spectral properties. Normally, a fluorophore will release light at its characteristic emission wavelength following excitation. When two suitable fluorophores are in proximity within the distance defined by the Förster radius, FRET will prevent fluorescent emission from the higher energy group. Instead, energy is transferred to the lower energy group, exciting the acceptor, and leading to fluorescence emission at a lower energy wavelength characteristic for the acceptor. Non-fluorescent acceptors exist which will accept energy from a donor without any resulting fluorescence emission. These acceptors as a group are known as "dark quenchers", and include Dabcyl, and BlackHole TM dyes.BlackHole TM dyes. 2) Donor and acceptor molecules must be in close proximity (typically 10-100 Å). FRET is a distance-dependent energy transfer between the electronic excited states of two dye molecules. The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined by the Förster radius (Ro). Competence and Service in Molecular Biology Real Time/Quantitative PCR

5. Fluorescence Resonance Energy Transfer - FRET - Diagram of the overlapping spectrum of a pair of FRET donor and acceptor dyes. Competence and Service in Molecular Biology Real Time/Quantitative PCR

6. TaqMan ® Chemistry 5´/3´ (reporter/quencher, I. e. Fam/Tamra) dual labeled oligonucleotide FRET for reporter-quencher distances up to 35 bases Free oligo does not give fluorescent signal Hybridization of probe to its complementary target - two- molecule conformation FRET is still working - no signal! TaqMan TM 5'-nuclease assay - physical separation of reporter and quencher Stimulated reporter (I. e. FAM at 488 nm) gives fluorescent signal (I. e. FAM at 520 nm) Degradation of probe during each amplification step Competence and Service in Molecular Biology Real Time/Quantitative PCR

7. R TaqMan ® Chemistry Competence and Service in Molecular Biology Real Time/Quantitative PCR

8. Competence and Service in Molecular Biology Real Time/Quantitative PCR Molecular Beacon ® Chemistry 5´/3´ (reporter/quencher) dual- labeled oligonucleotide Unimolecular stem-loop structure in the absence of targetstem-loop Internal hairpin brings the reporter and quencher groups in physical proximity FRET Conformational change by hybridization to its complementary target (two- molecule conformation) probe molecule unfolds and hybridizes in the presence of target reporter dye emits fluorescence signal

9. Molecular Beacon ® Chemistry Probe-target hybrids cannot co- exist with stem hybrids due to the rigidity of DNA helices. Perfect match probe-target hybrids are energetically more stable than the stem-loop structures. Mismatched probe-target hybrids are energetically less stable than the stem-loop structure. Extraordinary specificity Loop Sequence of 15 (high GC%) to 30 (low GC%) bases Sequence hybridizes to the complementary target sequence Stem 5-7 bases (Tm 55-70°C) 3´and 5´ends carry reporter/quencher Dyes Reporter: Fluo, Fam, Hex, Tet, Rox, Tamra, Cy3, Cy5,.. Quencher: BHQs, Dabcyl,... Competence and Service in Molecular Biology Real Time/Quantitative PCR