Transcript Slide 1

metabion´s history
Oligonucleotides –
Primers and Probes
by
… as quality counts!
Competence and Service in Molecular Biology
Real Time/Quantitative PCR
Fluorescence Resonance Energy Transfer
- FRET Principle of FRET
• 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).
• 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 S1 level of the donor to the
S1 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.
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Real Time/Quantitative PCR
Fluorescence Resonance Energy Transfer
- FRET Primary Conditions for FRET
1) Energy lost by de-excitation of the donor molecule, S1-S0, 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 molecule
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).
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 BlackHoleTM dyes.
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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
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 - twomolecule conformation
FRET is still working - no signal!
TaqManTM 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
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TaqMan® Chemistry
Polymerization
5'
3'
Forw ard
Primer
R
R = Reporter
Q
= Que ncher
Q
Pr obe
p 3'
5'
5'
Reverse
Primer
Strand displacement
3'
5'
Q
p 3'
5'
3'
5'
3'
5'
5'
Cleavage
R
Q
p 3'
5'
3'
5'
3'
5'
5'
Polymerization completed
5'
3'
5'
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R
Q
p 3'
5'
3'
5'
Real Time/Quantitative PCR
Molecular Beacon® Chemistry
• 5´/3´ (reporter/quencher)
labeled oligonucleotide
•
dual-
Unimolecular stem-loop structure
in the absence of target
Internal hairpin brings the reporter
and quencher groups in physical
proximity
FRET
• Conformational
change
hybridization
to
complementary
target
molecule conformation)
by
its
(two-
• probe molecule unfolds and
hybridizes in the presence of
target
reporter
dye emits
fluorescence signal
Competence and Service in Molecular Biology
Real Time/Quantitative PCR
Molecular Beacon® Chemistry
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
• Probe-target hybrids cannot coexist 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.
• Reporter:
Fluo, Fam, Hex, Tet, Rox,
Tamra, Cy3, Cy5, ..
• Quencher:
BHQs, Dabcyl, ...
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Extraordinary specificity