Data Analysis

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Slide1
Probe and Primer Design
Domestic teleconference 888-225-3022 Conference
Code 858 535 5400
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Primer / Probe Design and OptimizationPractical
considerations

• Primer Design
• Probe Design
• Dye and Quencher Selection
• Oligo Synthesis and Testing

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Target choice

• Target size 60-120 bp (200-400bp for SYBR)
• If using a cDNA template, span exon-exon
  junctions to avoid genomic DNA contamination.
• Try to avoid repetitive or highly structured
  regions (CpG islands and control regions at the
  5end tend to form complex structures).

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Primer properties

• 15-30 bp in length
• Primer pairs should have a theoretical Tm within
  2 C of each other
• Use the same target annealing temperature for all
  primers (to favor multiplexing in the future).
  Typically 55-60C.
• Taqman primers should usually have a Tm of at
  least 60C.
• 40-60 GC content (determined by Tm requirement
  and target choice). If target sequence requires,
  can try 20-80
• Avoid G/C clamps at the 3end to prevent primer
  folding on itself.
• If requested by the design software, use 100 mM
  monovalent cation and 5 mM Mg.
• Always BLAST your primers.

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TaqMan Probes
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Taqman Probe design

• 20-30 bp in length, Tm 10C higher than primers.
• 35-65 G/C more Cs than Gs. Can try as high as
  80 or as low as 20 if the region is
  particularly GC or AT rich.
• Avoid runs of 3 of the same nucleotide,
  especially Gs.
• 5 base ? G.
• When the probe and primers anneal to the target,
  the 5 end of probe should be 3 nucleotides from
  the 3 end of the primer on same strand (max of
  10-12).
• Test that primers and probe are not complementary
  to each other. (delta G free energy at 25C should
  be greater than -2)

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Molecular Beacons
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Molecular Beacon design

• Begin design as for a hydrolysis probe.
• Tm of probe region should be 7-10C above target
  annealing temp.
• To the chosen sequence add a stem
• 5-7 bp in length, with similar Tm
• as the probe region.
• Test folding using appropriate software (mfold).
• http//www.bioinfo.rpi.edu/applications/mfold/
• Check that there is no complementarity between
  primers and probe.
• Tm of probe alone and probe complement should
  be verified experimentally

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Molecular Beacon Melting Curve

• Properly designed Molecular Beacons can
  effectively discriminate between targets with a
  single bp mismatch.

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Primer/Probe Complementarity

• More negative deltaG (free energy) indicates
  dimer formation is more spontaneous event
• In general, the deltaG values should fall in the
  following ranges
• Singleplex reaction Greater than -2 for each
  oligo pair (more positive)
• Duplex reaction Greater than -4 for each oligo
  pair (more positive)
• Triplex reaction Greater than -6 for each oligo
  pair (more positive)
• Quadriplex reaction Greater than -8 for each
  oligo pair (more positive)
• Can calculate deltaG values for oligo pairs at
  http//www.bioinfo.rpi.edu/applications/mfold/
• using the 2-state hybridization server, with
  parameters at 25C hybridization temperature,
• 100 mM Na and 5 mM Mg
• Check 3 complementarity of primers by scanning
  sequences manually
• If you are using a primer design program like
  Primer 3 or Beacon Designer 2, the program
• will take the primer interaction into account, so
  you should not need to calculate deltaG.

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Which dye to choose? Avoiding Spectral Overlap
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Reporter-Quencher Pairs
We have heard favorable feedback from customers
regarding the companies IDT and Biosearch
Technologies for probe synthesis.
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Oligo Synthesis

• Primers
• Use desalted primers for sequence-specific
  detection. SYBR Green primers should be
  HPLC-purified.
• Aliquot stock solution of 20x (as determined by
  the optimization step), typically 6 - 18 µM each
  oligo.
• Test primers before ordering probe
• Probe
• Ensure matching fluorophore/quencher set.
• Double-HPLC purified (PAGE-purification also OK).
• Aliquot working stock at 2 - 4 µM (20x).

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Initial Primer Testing

• Primers
• PCR reaction and electrophoresis.
• SYBR Green run and dissociation analysis.

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Initial Probe Testing

• Probe
• analyze ?dR
• with Plate read
• (pre and post rxn.)
• Nuclease digestion (100nm probe in 25uL 1X buffer
  with 10U DNase or S1 _at_ RT 30 min.). Expect
  approx. 5000 counts or more difference between
  pre- and post-nuclease treatment.

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Good Probe Performance
Delta dRn 0.1 to 0.9
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Poor Probe Performance
Delta dRn 0.1 to 0.4
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QPCR Assay Optimization
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QPCR Reaction Components

• Primer concentrations
• 100-600 nM (start with 300 nM).
• 50-300 nM ( start with 150 nM) for SYBR Green.
• Probe concentrations
• 50-300 nM (start with 200 nM).
• Mg concentration
• 3.5-5.5 mM (start with 5 mM).
• 1.5-3.5 mM (2.5 mM) for SYBR Green.

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Primer Annealing Optimization

• There are two methods that can be used to
  optimize the annealing conditions for the
  primers.
• Alter the annealing temperature in the thermal
  cycling program. For this sort of optimization a
  QPCR instrument with a Gradient feature is
  useful. For multiplex assays, where the reactions
  are all run in the same tube this is generally
  not an option.
• The other option is to alter the relative
  concentrations of the primers. This method
  allows all the reactions to be run in the same
  tube at the same temperatures. This is the best
  method for multiplex reactions and also for
  optimizing Taqman reactions, which require a two
  step cycling with the annealing/extension step at
  60C.

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Assay Optimization

• Optimize primer concentrations first
• Test a matrix of forward and reverse primer
  concentrations against fixed concentrations of
  template, and Mg
• template concentration 5 - 10k copies (or 10ng)
  works well
• Mg concentration usually 5 mM (2.5 mM for
  SYBR)
• run all optimizations in triplicate and include
  one NTC
• confirm optimal primer pair with standard curve
• Second optimize probe concentrations
• Test different probe concentrations w/ optimal
  primer concentration
• confirm optimal probe concentration (and optimal
  primers) with standard curve
• Lastly optimize Mg concentration
• usually last step to attempt if assay is still
  not performing optimally
• if multiplexing, choose a set concentration for
  the assay

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Multiplex Primer Optimization Matrix

• Two Schemes

Optimize concentration of forward and reverse
primers and total primer concentration.
Optimize concentration of forward and reverse
primers with same total primer concentration.
(Taqman 600 nM total) (SYBR Green 300 nM
total) This scheme requires far fewer wells, so
it is generally preferred.
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Primer Matrix Results
The data from the complete primer matrix can be
plotted in a graph such as this.
The data from the abbreviated primer matrix can
be plotted in a graph such as this.
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Prim Titr
Primer Titration

• Goal
• Low Ct
• Lower concentrations (especially when
  multiplexing)
• Least variability between replicate runs
• Clean melt curves
• Next, determine efficiency of amplification by
  serial dilution standard curve in next run.

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Standard Curves
Standard Curves in Optimization
During assay design and optimization, it is very
useful to run a standard curve for each primer
and probe set, even if you do not plan to use the
standard curve method of quantification. The
standard curve is a good indication if further
assay optimization is necessary. To generate
this curve, use the optimized concentration of
primers from your primer matrix and perform a
serial dilution of template.
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Standard curves
Standard Curve

• You can then plot the Cts of your standards (Y
  axis) versus the log of your initial quantity (X
  axis) to generate a standard curve.
• The standard curve should be linear over the
  entire range of where you expect your unknowns to
  fall. How well these points fit to a straight
  line can be measured by the Rsq value.
• The slope of this line can be used to calculate
  the efficiency of amplification.

Efficiency 10(-1/slope) - 1
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Serial Dilution Std Curve
Rsq 0.996 Efficiency 96.3
Ct Range
6pt Std Curve over 3x orders dynamic range (Std
Curve Specifications Rsq gt 0.985 and Efficiency
90-110)
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Eff import
Why is Efficiency important?
To accurately correlate the amount of amplified
DNA to amount of initial target DNA,
amplification efficiency is required.
YX (1 E)n
Y PCR amplified quantity X target DNA
quantity prior to PCR E amplification
efficiency n number of cycles
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Factors that Influence Efficiency

• Length of the amplicon
• G/C content
• Secondary structure
• Concentrations of reagents
• Quality of reagents
• Inhibition (accumulation of pyrophosphates)
• Etc...

If proper assay optimization is performed, the
effect of these variables in the assay is
minimized and a quantitative result can be
obtained.
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Stratagenes Human QPCR Reference RNA

• Can be used as template to optimize primer/probe
  sets
• Reliable source of total RNA pooled from 10 cell
  cultures (representing different tissues) to
  represent the majority of human genes
• Avoid wasting precious RNA validate immediately
• Gives realistic amplification efficiency
• Can verify that primers dont cross react
• Use same reference for all assays
• Undetectable gDNA (double DNase treated)

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Advantages of Multiplexing

• Mutliplex vs. Independent reactions.
• Target Internal control/Normalizer can be
  easily multiplexed.
• If you plan to perform a very large number of
  runs with the same sets of primers and probes
  (for example, if you are setting up a screening
  assay) the extra design time required for a 3 or
  4-way multiplex is justified savings from running
  fewer samples over time.
• Save setup time and reagent cost (Taq)
• Can get data more quickly because fewer runs have
  to be performed.

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Multiplex Assay Design

• Primer and Probe Design
• Primers and probes are designed the same for
  multiplex reactions as with simplex reactions.
• All amplicons should be similar length, G/C
  content, and Tm.
• Extra care should be given to ensure that all
  oligonucleotides have minimal interference with
  one another.
• assays designed from the very beginning as
  multiplex assays tend to produce better results
  than a combination of multiple simplex assays
• Using lower primer concentration minimizes the
  possibility of oligo-oligo interaction
• high amplicon concentrations can shorten the
  dynamic range of a multiplexed assay
• Probe Labels
• Choose fluorescent dyes that maximize multiplex
  performance
• Dyes should be used that have minimal spectral
  overlap
• signal cross-talk will decrease the dynamic range
  of a multiplex assay
• Choose the best quencher for each dye Black Hole
  Quenchers
• FAM, HEX, ROX, and Cy5 are the best combination
  to use in 4-plex assays

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Multiplex Assay Validation

• Determine the efficiency of amplification
• First, create a dilution series of template for
  each target (ex 4-5pt standard curve in
  triplicate that covers 2-3 logs of concentration)
• Test each reaction independently and multiplexed
• Determine the slope of each standard curve
• From this derive the efficiency of amplification
  for each assay
• Determine if the amplification efficiency of each
  multiplexed reaction differs significantly from
  the independent reaction
• Efficiency difference should not be greater than
  5
• Determine the sensitivity and dynamic range of
  the assay
• Multiplex and independent reactions should be
  within 1 Ct

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Singleplex and Multiplex Std Curve
Mutliplexed (4)
Independent
Efficiency of both Std Curves within 5 and Ct
values within 1 Ct
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Multiplex Reaction Components

• Performing 3-plex or 4-plex assay it is necessary
  to increase reaction component concentration
• Increase Taq concentration (50-100).
• Increase dNTP concentration (50-100).
• Increase Mg concentration (increase by
  0.25-0.50 mM).
• In some cases, increase buffer concentration to
  1.5x.
• Some 2-plex assays also work much better with
  increased reactions components, typically a good
  approach if independent and multiplex reaction
  Cts are slightly off

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QPCR Standard Curve FAM, HEX, CY5, Texas Red
Simplex Assays
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QPCR Standard Curve FAM, HEX, CY5, Texas Red
Multiplexed Assay

• CY5 primer/probe changing slope.
• In multiplex, slope indicates 180 efficiency,
  indicating some sort of interaction with other
  reactions to generate additional signal

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Assay Troubleshooting

• Still cant multiplex?
• Determine the problem oligo by systematically
  removing oligos until the problematic one is
  found
• redesign the oligo(s) to be compatible
• If problem target is dominant in the reaction,
  can attempt to primer limit the target.
• Minimize dR without changing Ct of dominant target

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Thank You!
If you have any questions, please email us at
qpcrsystemssupport_at_stratagene.com