Fluorescent reporter molecules are used with various molecular technologies to monitor biological processes.

1. PRIYANKA GUPTA
PhD Research Scholar
Dept. Of Lab. Medicine ,
AIIMS, New Delhi02-03-2016 1

2.  Fluorophore is a fluorescent
chemical compound that can
re-emit light upon light
excitation.
 Principle of fluorescence
given by “Aleksander
Jablonski” named as
Jablonski energy diagram.
Introduction
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3.  They are sometime used alone, part of an enzyme or
covalently bonded with bioactive molecules. (e.g.
antibodies, protein & nucleic acid)
 Fluorophores are notably used to stain tissues, cells, or
materials in a variety of analytical methods, i.e., fluorescent
imaging and spectroscopy.
 Fluorescent reporter molecules are also used with various
molecular technologies to monitor biological processes for
example:
1. PCR (Polymerase Chain Reaction)
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4. 2. Real time PCR
3. Flow cytometry
4. LAMP (Loop Mediated Isothermal Amplification)
5. Fluorescence In Situ Hybridization (FISH)
6. DNA Microarray
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5. Acridine 362 462
Caumarine 432 472
Cy3 552 570
Cy5 646 667
EtBr 493 620
Fluorescein 495 520
FAM 494 525
TAMRA 565 580
ROX 587 607
Texas Red 583 603
TET 521 536
JOE 528 554
HEX 535 556
Light cycler 610 590 610
Light cycler 640 625 640
Oregon Green 500 521
Rhodamine 503 580
VIC 538 554
Alexa Fluor
species
varies varies
Yakima yellow 530 549
Dye Absorption
(nm)
Emission
(nm)
Dye Absorption
(nm)
Emission
(nm)
Fluorophores
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6. 02-03-2016 6
Fluorophore Dyes used in Flowcytometry:

7.  Mostly used fluorophore dyes in DNA microarray is
Cyanine dyes e.g. Cy3, Cy5, etc.
 Alexa fluore dyes are generally used in Fluorescence In Situ
Hybridization.
 In the real time PCR the fluorophore molecules are
categorised into two types:
1. Donor or Reporter molecule : Fluorescent molecule, emit
energy as fluorescence. e.g. FAM, JOE, etc.
2. Acceptor or Quencher molecule : The acceptor may or
may not fluorescent molecule. When it is non fluorescent it
is quencher, the electronic energy of quencher is dissipated
in the form of heat. e.g. BHQ-1, BHQ-2, Dabsyl, etc.
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8. Reporters and quenchers for real time PCR:
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9. Donor and Acceptor for real time PCR:
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10. Ethidium Bromide (EtBr) :
 Ethidium bromide is a
heteroaromatic cationic dye.
 Ethidium bromide is the most
commonly used dye for DNA
and RNA detection in gels.
 Ethidium bromide intercalate
between the base pairs of
double helix DNA or RNA.
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Fluorophore dye for PCR

11.  Ethidium bromide has
UV absorbance at 493 nm
and emission at 620 nm.
 Ethidium bromide is a
powerful mutagen,
extremely toxic by
inhalation, ingestion and
skin contact, and a
suspected carcinogen and
reproductive toxin.
Agarose gel with UV illumination, stained
by EtBr
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12. Types of Fluorescence based
Chemistries
DNA Intercalating dyes Fluorophore labeled oligonucleotides
SYBER Green 1
SYTO
BEBO
BOXTO
PNA
Hyb Probe or FRET
TaqMan
Cyclicons
Scorpion
Molecular Beacon
LNA
LUX
Probes
Primer-probes
NA analogues
EvaGreen
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13.  These are the intercalating dyes.
 Bind with the minor groove of dsDNA and leads to
fluorescence emission.
 The most common examples of these dyes is SYBER Green 1.
 The use of these dyes allow the detection of specific products,
nonspecific products and primer-dimer produced during the
reaction.
DNA Binding Dyes
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14. SYBR Green I
 It is asymmetrical cyanine dye with two positive charges.
 It absorbs blue light at 497 nm and emits green light at 520
nm.
 Bind with minor groove of DNA. This dye is useful to
perform melting curve analysis.
 Melting curve analysis is used for discriminating two or
more different DNA sequence in a single PCR reaction of
multiplex assays.
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15.  Applications:
1. Pathogen detection.
2. Gene expression.
3. SNP detection
4. GMO detection.
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16.  These are small fluorescent molecules attached to
oligonucleotides to be used function as probe.
 Two types of fluorophores: Donor or reporter
Acceptor or quencher.
 The transfer of excited state energy from a donor to acceptor
use FRET (Fluorescence Resonance Energy Transfer)
mechanism.
 Two different types FRET mechanism use:
Fluorophore-labeled Oligonuleotide
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17. 2. FRET : Transfer energy emitted as fluorescence because the
acceptor molecule is fluorescent.
1. FRET-quenching : Electronic energy of the quencher is
dissipated as heat because quencher is a nonfluorescent molecule.
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18. Probes
 Holland et al. (1991)
 These probes are oligonucleotides containing a reporter
fluorescent moiety at the 5’-end and an quencher at 3’ end.
 These are designed to bind to a specific region of the target
DNA.
 In solution the fluorescent signal is quenched due to the
FRET-quenching phenomenon.
1. TaqMan or Hydrolysis probes:
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19.  In the extension phase , the
bound hydrolysis probe is
removed by the 5’-3’
exonuclease activity of DNA
polymerase and reporter dye
generate florescence.
Applications :
1. Use in singleplex & multiplex
format for viral detection .
2. Viral/ bacterial load
quantitation.
3. Genotyping.
4. Gene Expression.
5. Mutation detection.02-03-2016 19

20. 2. Hyb Probe or FRET :
 Heller and Morrison. (1985)
 These probes consists a pair of oligonucleotides bind to
adjacent target DNA sequences.
 The first probe carries a donor fluorophore at its 3’ end and the
second probe contain acceptor fluorophore at its 5’ end .
 These probes hybridize to the target DNA sequences in a
head-to tail combination bring the fluorophore into close
proximity, producing fluorescence due to FRET mechanism
in annealing phase.
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21. Applications :
1. Use in singleplex & multiplex format for viral detection .
2. Viral/ bacterial load quantitation.
3. Microarray validation.
4. Gene Expression.
5. Mutation detection.
6. Melting curve analysis.
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22. 3. Molecular Beacon probes :
 Tyagi and Kramer. (1996)
 They are single standard hairpin
shaped oligonucleotide probes which
contain :
1. one loop, 18-30 bp complementary to
the target DNA sequence
2. a stem contain two complementary
sequences at end of the probe .
3. a reporter dye at 5’ end and
quencher at 3’ end.
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23.  During the annealing phase, the
molecular beacon probe unfolds and
binds to the target DNA sequence,
leading to fluorescence emission.
Applications :
1. Use in singleplex & multiplex
format for viral detection .
2. Viral/ bacterial load quantitation.
3. Microarray validation.
4. Gene Expression.
5. Mutation detection.
6. Melting curve analysis.
7. Allelic discrimination.
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24. Primer-probes
 Primer- probes are oligonucleotides that combine a
primer probe in a single molecule.
 These are nonspecific, so melting curve analysis to
determine the efficiency of the reaction is recommended.
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25. 1. Scorpion Primer-probes
 Whitcombe et al. (1999)
 The hairpin structure of scorpion :
1. a reporter at 5’-end
2. an internal quencher at the 3’-end
3. 3’ end attached to the 5’-end of HEG
(hexa glycol) blocker.
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26.  After probe binding to target, DNA polymerase amplify the
target sequence. In the next denaturation, the specific
sequence of the probe bind to the complementary region
within same strand of newly amplified DNA, leading to
fluorescence emission.
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27.  It is an inexpensive system in which primer-probe combines
and detection mechanism in the same molecule.
 Applications :
1. Pathogen detection in singleplex & multiplex format.
2. Viral/ bacterial load quantitation.
3. Genotyping.
4. Mutation detection.
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28. 2. LUX ( Light-Upon-Extension)
 Nazarenko et al.
 The 3’-end act as primer and contain a single reporter located in
the guanosine rich region of the primary sequence.
TM
The hairpin structure confer the
ability to decrease the fluorescence
signal when the primer-probe is
free. Maximum fluorescence
emission takes place when it
incorporate with dsDNA during
extension phase.
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29. Applications :
1. It can be used for pathogen detection in singleplex &
multiplex format.
2. Viral/ bacterial load quantitation.
3. Genotyping.
4. Mutation detection.
5. Gene expression analysis.
6. GMO detection.
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30. 3. Cyclicons
 Kandimalla & Agrawal. (2000)
 It contain a long primer-probe
complementary to target sequence
and a short modified oligo attached
through 5’-5’ ends. Which binds to 3’-
end of the primer-probe forming
cyclic structure with two 3’-ends.
Cyclicons have a reporter at free 3’ –
end and a quencher at 5’-end.
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Quencher
Reporter
3’
Cyclicon primer-probe
FRET

31.  It binds to the complimentary sequence the cyclic structure
opened up and the fluorophores are separated far enough to
disrupt FRET-quenching resulting fluorescence emission.
Applications :
1. It can be used for pathogen detection in singleplex &
multiplex format.
2. Viral/ bacterial load quantitation.
3. Genotyping.
4. Mutation detection, SNP detection.
5. It can be directly fixed to solid supports o the chips for
high-throughput screening in solid-phase PCR.
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32. Nucleic acid Analogues
 Nucleic acid analogues are compounds that are analogous
to naturally occurring RNA and DNA.
 e.g. PNAs , LNAs and ZNAs, etc.
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33. 1. PNAs:
 Nielson et al.
 They are achiral and electrically
neutral DNA analogues in
which the sugar-phosphate
backbone has been replaced by
a peptide of N-(2-aminoethyl)-
glycine units.
 PNAs able to interact with either dsDNA or RNA with higher
affinity and greater specificity than conventional
oligonucleotide.
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34.  The mechanism of primer-probes or probes in which PNA
molecules have been introduced is identical to the method of
action of conventional probes.
 Applications:
1. Induce DNA recombination or block PCR amplification of
specific gene.
2. Uniquely, allelic discrimination of single nucleotide
polymorphism can be accomplished by using PNA
molecular beacon
3. Discrimination between DNA and cDNA sequence in
prokaryotes.
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35. 2. LNAs:
 Wengel and co-workers in 1998.
 LNAs are DNA or RNA sequence in a conformation that
contain one or more modified nucleotides.
 They have a methylene bridge between atoms 2’-O and 4’-C in
the ribose ring to form a bicyclic ring.
 LNA containing primer-probes or probes exhibit the same
mode of action as that of convention probes.
 The use of LNA Molecular beacon and LNA TaqMan probes
has been reported for SNP detection, GMO detection and viral
quantification.
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