Probe designing in
Real Time PCR
Parameter for designing
Taqman probes
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1. Tm criteria: The primer melting temperature (Tm)
should be around 58-60°C, and TaqMan® probe Tm
should be 10°C higher than the Primer Tm. The Tm of
both the primers should be equal
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2. Length criteria: Primers should be 15-30 bases in
length
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3. GC content: The G+C content should ideally be 3080%. If a higher G+C content is unavoidable, the use of
high annealing and melting temperature co-solvents such
as glycerol would be deemed essential
Parameter for designing
Taqman probes
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4. GC clamp: The total number of Gs and Cs in the last
five nucleotides at the 3' end of the primer should not
exceed two. This helps to introduce relative instability to
the 3' end of primers to reduce non-specific priming
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5. Amplicon length: Maximum amplicon size should not
exceed 400 bp (ideally 50-150 bases)
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6. Runs and repeats: The probes should not have runs of
identical nucleotides (especially four or more consecutive
Gs), G+C content should be 30-80%, there should be
more Cs than Gs, and not a G at the 5' end
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7. Genomic DNA avoidance: False-positive results are
obtained due to amplification of contaminating
Taqman probes applications
1. Quantitative real time PCR.
2. DNA copy number measurements.
3. Bacterial identification assays.
4. SNP genotyping.
5. Verification of microarray results
Parameter for designing
Molecular Beacon
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to detect the synthesis of products during polymerase
chain reactions, any region within the amplicon that is
outside the primer binding sites can be selected
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In order to discriminate between amplicons that differ
from one another by as little as a single nucleotide
substitution, the length of the probe sequence should be
such that it dissociates from its target at temperatures 710 ˚C higher than the annealing temperature of the PCR

If single-nucleotide allele discrimination is not desired,
longer and more stable probes can be chosen
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Loop: This is the 15-30 base pair region of the molecular
beacon which is complementary to the target sequence
Parameter for designing
Molecular Beacon
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ensured by choosing a stem that melts 7-10˚C higher than
the annealing temperature of the PCR
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Stem: The beacon stem sequence lies on both the ends of
the loop. It is typically 5-7 bp long at the sequences at
both the ends are complementary to each other
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have a very high GC content (75 to 100%)
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DNA folding program, such as the Zuker DNA folding
program available on the internet at
http://frontend.bioinfo.rpi.edu/applications/mfold/cgibin/dna-form1.cgi, is utilized to estimate the melting
temperature of the stem
Parameter for designing
Molecular Beacon
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In general, 5 basepair-long GC-rich stems will melt between
55 and 60˚C,
6 basepair-long GC-rich stems will melt between 60 and 65˚C,
and
7-basepair long GC-rich stems will melt between 65 and 70˚C
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Do not use G residues near the end to which the fluorophore
(5’) is attached (instead, use them at the end where the
quencher (3’) is attached), G residues tend to quench the
fluorophore
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Small stems within the probe's hairpin loop that are 2- to 3nucleotides long do not adversely effect the performance of
molecular beacons.
Parameter for designing
Molecular Beacon
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no regions of substantial complementarity that may cause the
molecular beacon to bind to one of the primers
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amplicons should be less than 150-basepairs long
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Molecular beacons are internal probes that must compete with
the other strand of the amplicon for binding to the strand that
contains their target sequence
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Having a shorter amplicon allows the molecular beacons to
compete more efficiently, and therefore produces stronger
fluorescence signals during real-time PCR
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smaller amplicons result in more efficient amplification
Parameter for designing
Molecular Beacon
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the magnitude of the molecular beacon signal can be increased
by performing asymmetric PCR, in which the primer that
makes the strand that is complementary to the molecular
beacon is present at a slightly higher concentration than the
other prime

Molecular beacons can also be designed with the help of a
dedicated software package called 'Beacon Designer,' which is
available from Premier Biosoft International
(www.premierbiosoft.com)

A detailed description on the design, synthesis and application
of molecular beacons used for genotyping single nucleotide
polymorphisms appeared in

Marras SAE, Kramer FR, and Tyagi S (2003) Genotyping single nucleotide polymorphisms with molecular beacons. In Kwok, P. Y.
(ed.), Single nucleotide polymorphisms: methods and protocols. The Humana Press Inc., Totowa, NJ, Vol. 212, pp. 111-128
Vet, J.A.M. and Marras, S.A.E. (2004) Design and optimization of molecular beacon real-time polymerase chain reaction assays. In
Herdewijn, P. (ed.), Oligonucleotide synthesis: Methods and Applications. Humana Press, Totowa, NJ, Vol. 288, pp. 273-290

Molecular Beacon applications
1 . SNP detection
2 . Real-time nucleic acid detection
3 . Real-time PCR quantification
4 . Allelic discrimination and identification
5 . Multiplex PCR assays
6 . Diagnostic clinical assays
Parameter for designing
Scorpion probes

Amplicon length: The primer pair should be designed to
give an amplicon of approximately 100-200 bp

Secondary structures: The designed primers should be
tested for hairpins and secondary structures. Ideally the
primers should have as little secondary structure as
possible

Tm criteria: The Tm's of the two primers should be
similar. Also, the stem Tm should be 5-10ºC higher than
the probe Tm
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Complementary probe: The scorpion® should be
written as the reverse complement of the target
Parameter for designing
Scorpion probes

Length criteria: Probe sequences should ideally be about
17-27 bases Also, the probe target should be 11 bases or
less from the 3' end of the scorpion®

Probe stem: The stem sequence can be of 6 to 7 bases,
mostly Cs and Gs, avoiding motifs. The 5' stem sequence
should begin with a C as G may quench the FAM

Primer: Probe hybridization: There is always the
possibility of the primer hybridizing to the probe element,
this will lead to leniarization of the probe in an
amplification-independent manner causing significant,
target-independent fluorescence
Scorpion probes applications
1. SNP analysis
2. Real-time PCR
3. Allelic discrimination
4. Single tube genotyping assay
Parameter for designing
SYBR Green primer

Careful primer design is critical

If primer dimers or other non-specific PCR products form,
they will incorporate SYBR Green dye

This may lead to inaccurate quantification, especially
when detecting sequences of low abundance

Amplicon length: The primer pair should be designed to
give an amplicon of approximately 250-400 bp

SYBR Green dye fluorescence depends on the presence
of double-stranded DNA, so products shouldn't be too
short

G/C content should be 20-80%
Parameter for designing
SYBR Green primer
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Template regions with obvious secondary structures or long
runs of the same nucleotide should be avoided when
designing primers as this hinders the primers from annealing
and extension

Avoid a 3' terminal T on primers if possible. Thymidine tends
to misprime more readily than other bases

Tm criteria: The Tm of 60-65 °C will be sufficient

BLAST your primer sequences. Run a similarity search of
primers against whole organism/non-redundant databases to
determine if primers might anneal to other (unwanted) targets

Run positive and negative controls with conventional
endpoint PCR
Choosing a Flourophore & Quencher
Fluorophores:
FAM
TET, VIC
HEX
JOE
CY3
TAMRA
CY3.5, Redmond Red
Texas Red, ROX
CY5 LC640
CY5.5 LC705
Quenchers:
BHQ-1
DABCYL
Eclipse
TAMRA
QSY-7
BHQ-2
BHQ-3
Choosing a Flourophore & Quencher
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BHQ3 Currently not available. Unstable. They are working on the
chemistry
DABCYL Good quencher for beacons not TaqMan probes.
Requires CLOSE proximity for quenching
QSY7 Good quencher but difficult to make
Eclipse Dark quencher from Synthetic Genetics. Similar to
DABCYL, may provide better results for Tex Red and ROX
Vendors:IDT is the most reliable in terms of quality of probes.
However, turn around time is often 1 week
NOTE: HEX/TAMRA as reporters: Choose BHQ1 or 2. HEX and
TAMRA can be difficult to purify but IDT does it well.
CY5: The best quencher would be BHQ3 but currently unavailable
due to the instability of the chemistry. So R&D is using BHQ2 now
Redmond Red is from Synthetic Genetics
Choosing a Flourophore & Quencher
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List of dye and quencher
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TaqMan Probe – FAM and TAMRA (higher background)
FAM and BHQ1 or BHQ2 (heat transfer, not light,
low backgroud, suitable for multiplexing)
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Molecular Beacons – FAM and DABCYL
FAM and BHQ1
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Scorpion – same as Molecular Beacons
Choosing a Flourophore & Quencher
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Multiplexing – choose dye that are spectrally distinct from each other
as possible or have little spectral overlap. Add in one reference dye
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Duplex reaction – FAM and HEX (JOE/VIC)(ROX as reference dye)
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Triplex reaction – FAM, HEX (JOE/VIC) and CY5 (ROX as
reference dye)
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Quadriplex reaction – FAM, HEX (JOE/VIC), CY5 and Texas Red
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Quencher should be dark quencher, even thought TAMRA is an
effective quencher, but emission spectra for TAMRA do have some
overlap with other dyes like HEX
Real Time primer/probe
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Use desalted primer with probe-based detection chemistry
If the primer are to be used with the SYBR Green I detection, it need
to be HPLC purify
Sequence specific probe should be double HPLC purified or PAGE
purified
Aliquot the primer or probe before storing at -20°C
Multiple freeze/thaw cycles can damage oligos, probes especially
fluorescent tags
Primer – not more than 20 times, for probe – not more than 5 times
Real Time primer/probe
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1.
2.
3.
4.
5.
Simple test can be performed to ensure that the probe is
quencher properly
Read the fluorescence from an aliquot of a probe
Digest 100nM of probe with 10U DNase or S1 nuclease in 25ul 1X
buffer
Incubate at room temperature for 30 min
Take a second fluorescence reading
The reading should be increase >5000 counts in raw fluorescence
signal (digestion free the fluorophore)
Thank you