Overview of Real Time PCR

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Overview of Real -Time PCR

• Real-Time PCR combines DNA amplification with
  real time amplified product detection in a single
  tube.
• More specific then gel or hybridization assays.
• Less time consuming
• Quantitative results as opposed to
  semi-quantitative or qualitative results.
• Human (higher primate) or Y chromosome specific
• Monitors for PCR inhibition

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Introduction

• Methods based on changes in fluorescence
  proportional to the increase in either specific
  or non-specific PCR product. The fluorescence is
  monitored during each PCR cycle to provide an
  amplification plot allowing the user to follow
  the reaction.

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Amplification Plot
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High Throughput Systems

• ABI Prism SDS 7000
• ABI Prism SDS 7700
• ABI 7300 and 7500 Real-Time PCR Systems
• iCycler iQ
• Mx 3000p Multiplex quantitative PCR system

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ABI Prism SDS 7000

• 96 well format
• Illuminated by a tungsten-halogen lamp
• 4 optical filters
• Optimized for FAM, SYBR green I, VIC, Yakima
  Yellow, TAMARA and ROX dyes
• Multiplexing capabilities
• Time 2 hours
• Display includes disassociation curve, and
  results during the run

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ABI Prism SDS 7700/7900

• 96 well format.(7900 has 384 well format and
  robotic capability).
• Excitation is done by a laser.
• Fluorophores with an emission between 500 and 660
  nm can be used. Includes FAM, TET, JOE, Yakima
  Yellow, VIC, ROX, TAMARA, SYBR green I.
• 2 hour run time.

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ABI 7300 and 7500 Real-Time PCR Systems

• 96 well plate format
• Excitation by Tungsten-Halogen lamp
• 25-100 µl reaction mix.
• 7300- FAM, SYBR green I VIC, Yakima Yellow, or
  JOE, TAMARA, ROX
• 7500- 5 color detection (NED, Cy3, Texas Red,
  Cy5) better sensitivity with longer wavelengths
• 2 hour run time

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iCycler iQ

• BioRad
• 96 well format
• PCR machine with an optical unit
• Halogen light source
• CCD camera measures emission from each well
  simultaneously and detects 4 colors at once (FAM,
  SYBR green I, VIC, Yakima Yellow, HEX, TET, Cy3,
  Cy5, Texas Red, TAMARA, ROX
• 2 hour run time

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Mx3000p Multiplex quantitative PCR system

• Stratagene
• Detects 4 fluorophores w/in the same reaction
  using four different channels (customized
  filters). Multiplexing ( 4 different targets at
  the same time).
• 96 well format
• Halogen lamp
• 350-750nm
• Detection- Scanning photomultiplier tube (PMT)
• Dyes- FAM, SYBR green I, TET, HEX, JOE, VIC,
  Yakima Yellow, TAMARA, Cy3, Cy5, Texas Red, ROX,
  Alexa Fluor 350
• Run Time 350

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Flexible Instruments

• LightCycler 2.0 Instrument
• Smartcycler
• RotorGene 3000 Four Channel Multiplexing System
• Used for multiplexing and performing multiple
  experiments each with a different set of
  parameters at the same time.

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Quantitative and Qualitative PCR Technologies

• Two types of Real-Time PCR detection chemistries
• 1. Specific Sequence Detection- Distinguishes
  between a specific sequence of interest and
  non-specific products. Can be used to detect
  different alleles.
• 2. Non-Specific Detection- Detects any dsDNA
  produced during the reaction

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Non-Specific Detection Systems

• Uses a dsDNA intercalating dye that that
  fluoresces once bound to DNA. The most common is
  SYBR green 1. Excites at 497 nm and emits at 520
  nm.
• Do a melting curve start at 40C-95C. The
  fluorescence will decrease when the dsDNA
  disassociates. This depends on length and bp
  composition. If the PCR reaction is optimized
  will get a single peak corresponding to the
  proper primer pair. Can be useful in quantifying
  DNA.
• Useful in optimizing a PCR reaction before
  developing more expensive specific tests.

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Specific Detection Systems

• TaqMan Probes
• LNA Double Dye Oligonucleotide probes
• Molecular Beacon Probes
• Scorpions primers

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TaqMan Probes

• Double- Dye Oligonucleotides or dual labeled
  probes.
• Method of choice for forensic DNA quantitation
• Consists of a ssDNA probe that is complemenatry
  to one of the amplicon strands
• A fluorophore (FAM) is attached to one end of the
  probe and a quencher (TAMARA) and a quencher to
  the other end. The fluorophore is excited my the
  instrument and passes its energy via FRET to the
  quencher.

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FRET and NFQ

• Fluorescent Resonance Energy Transfer
• NFQ- Non- Fluorescent Quencher

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FRET Technology

• High energy fluorophore (reporter dye 6FAM) is
  close to the NFQ and the energy will transfer
  from high to low. As PCR products are formed the
  specific TaqMAn probe will anneal to its
  complementary sequence in the amplicon. Taq
  Polymerase (due to its 5-3 nuclease activity)
  will cleave the probe separating the fluorophore
  (reporter) dye from the quencher. The
  fluorescence is no longer quenched and is
  proprotional to the number of amplicons produced.

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TaqMan Probe Mode of Action
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Uses for TaqMan Probes

• DNA Quantitation
• Mutation Detection-Probe designed to hybridize
  over mutation
• Allelic Discrimination
• Gene Expression
• Can be multiplexed
• Dark Quenchers- absorb emitted energy, but
  release it as heat as opposed to fluorescence

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LNA Double Dye Oligonucleotide Probes

• LNA- Locked Nucleic Acids
• Integrating LNA bases into the DNA duplex of the
  double dye oligonucleotide probes will change the
  conformation from the B to the A form and
  increase its stability, thus increasing its TM
  value resulting in shorter, more specific probes.
• Useful in applications requiring high affinity
  probes such as SNP detection, gene expression
  profiling and in situ hybridization.

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LNA Bases

• LNA is a bicyclic RNA analogue in which the
  ribose moiety is structurally constrained by a
  methylene bridge between the 2- oxygen and 4-
  carbon atoms. When these LNA bases anneal with
  the DNA bases they cause a conformational change
  to occur in the helix.

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LNA Base Structure
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B and A Helices
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Molecular Beacon Probes

• Consist of a hairpin loop where the loop is a
  single- stranded probe that is complementary to
  the amplicon. The stem is usually 6 bases long
  and consists mostly of C and Gs. It holds the
  probe in the hairpin structure. A fluorphore is
  attached to one of the stem and a quencher the
  other. When the amplicon is produced during PCR,
  the probe will bind to its specific target
  sequence provided the probe-target duplex is
  thermodynamically more stable then the hairpin
  loop at the fluorescent acquisition temperature.
  After the probe binds to its target the hairpin
  is opened and the fluorophore and quencher are
  separated.

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Continued

• The increase in fluorescence is reversible
  because until the temperature is high enough the
  hairpin loop will reform. This can produce a
  melting curve that is useful for observing the
  dynamics of the reaction and determining the best
  temperature for fluorescence acquisition.

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Diagram of a Moleucular Beacon Probe
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Scorpion Primers

• PCR Primers with a stem-loop tail containing a
  fluorophore and a quencher. The tail is separated
  from the primer by a PCR blocker,a chemical
  modification that prevents Taq from copying the
  stem-loop tail of the Scorpion primer. This
  prevents non-specific opening of the loop. The
  PCR blocker links the loop to the 5 end of the
  primer. The primer extends during the PCR
  amplification and the specific probe sequence
  will bind to its complement sequence. The hairpin
  loop opens up during hybridization and the
  fluorescence is no longer quenched. Because
  enzymatic cleavage is not required the time it
  takes to detect fluorescence is decreased. Main
  advantage to TaqMan probes is low background and
  fast reaction mechanism. Binding is more specific
  then TaqMan probes and can produce a melt curve
  analysis like Molecular Beacon probes. They can
  be used in DNA quantitation, allelic
  discrimination and mutation detection. Capable of
  multiplexing.

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Scorpion Primer Reaction Diagram
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Technology Compatibility
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Fluorophore and Quencher

• The choice of which fluorophor and quencher
  combination to use will give different results in
  terms of sensitivity.
• Fluorophore- a molecule that emits light of a
  certain wavelength after having absorbed light of
  a specific, but different wavelength first. The
  emission wavelength is always higher than the
  absorption wavelength. The fluorophore absorbs
  light energy and is promoted to an excited state,
  when it falls back to its ground state the excess
  energy is released.
• Quencher- a molecule that accepts energy from a
  fluorophore in the form of light and disapates
  this energy either in the form of light or heat.

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Fluorophore and Quencher Structures

• Flourophore
• Quencher

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Fluorescence Principle
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Proximal and FRET Quenching

• Proximal- Fluorophore is in close proximity of
  the quencher energy is transferred from
  fluorophore to quencher and dissipates as heat
  w/o fluorescence. Also called collisional
  quenching. Dark Quenching.
• FRET Quenching- The fluorophore transfers its
  energy to the quencher (which can be another
  fluorophore) the energy is released as
  fluorescence of a higher wavelength. The
  efficiency is related to the distance (more
  precisely the Forster distance 1/r6, where r
  the fluorophore distance) between the fluorophore
  and quencher. TAMARA

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Proximal and FRET Quenching Diagram

• Proximal
• FRET

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Optimal Fluorphore Quencher Combination

• The absorption spectrum of the quencher needs to
  have good overlap with the emission spectrum of
  the fluorphore to achieve optimal quenching.
  Quenchers have a quenching capacity throughput
  their absorption spectrum, but work best close to
  their absorption maximum.

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Efficient Quenching of Fluorphores

• If the fluorescence is to high due to incomplete
  quenching the relative fluorescence signal
  (signal to noise ratio) will decrease. The
  fluorophore-quencher combination which gives the
  highest signal to noise ratio should be selected.

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ROX Passive Reference

• Used by ABI
• Correct for pipetting inaccuracies
• Does not participate in PCR
• Provides an internal fluorescent reporter to
  which the reporter fluorescent can be normalized

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Rn and Bn

• Rn (normalized reporter signal) reporter
  signal/ROX passive reference signal
• This corrects for pipetting error only, not
  background.
• ?Rn is used to correct for background.
• ?Rn Rn Bn
• Bn (normalized baseline) baseline signal/ROX
  passive reference
• ?Rn (reporter signal/ROX passive reference
  signal)- (baseline signal/ROX passive reference
  signal
• Thus the ROX passive reference signal influences
  both the Rn and Bn

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Ct Value

• Ct value- The cycle in which a significant
  increase in reporter signal above threshold can
  be detected, i.e. the cycle in which the growth
  curve crosses the threshold.
• The automatic threshold is calculated by the
  software and is defined as (average baseline
  plus) (standard deviation of the baseline)
• The ROX reference influences the baseline and
  noise level. When the level of ROX increases, the
  noise will decrease as will the ?Rn. It is
  therefore important to determine the right level
  of ROX that does not compromise ?Rn.
• The Ct value is directly dependent on the
  threshold level and indirectly dependent on
  baseline level and ROX reference level. Minor
  variations in ROX level can change Rn, Bn, ?Rn,
  threshold and Ct.
• Use a Master Mix that already contains ROX

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Quantitative PCR

• Quantitative PCR software uses the exponential
  phase of PCR for quantitation. PCR is initially
  an exponential process but reaches a plateau
  phase as one or more of the reaction reagents
  become limited. Reactions can plateau at
  different levels even if they have the same
  starting concentration of target. During the
  exponential phase the amount of target is assumed
  to be doubling every cycle w/o any bias from
  limiting reagents. Analyses use the Ct value, the
  point cycle number, at which the signal is
  detected above the background and the
  amplification is in the exponential phase. The
  samples with the most abundant amount of DNA
  template reach the Ct value earlier than the
  samples with less starting DNA. The results are
  normalized by comparing them against a standard
  curve developed by running a series of known DNA
  standards each of which has a different DNA
  concentration.

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Caution Notes

• 1. DNA concentration results are dependent on
  Molar Ratios and Molar Concentrations. When
  comparing results, make sure that the reaction
  volumes are the same.
• 2. Be wary of comparing results from different
  instruments or different platforms