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Overview of Real Time PCR

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Title: Overview of Real Time PCR


1
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

2
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.

3
Amplification Plot
4
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

5
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

6
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.

7
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

8
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

9
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

10
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.

11
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

12
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.

13
Specific Detection Systems
  • TaqMan Probes
  • LNA Double Dye Oligonucleotide probes
  • Molecular Beacon Probes
  • Scorpions primers

14
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.

15
FRET and NFQ
  • Fluorescent Resonance Energy Transfer
  • NFQ- Non- Fluorescent Quencher

16
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.



17
TaqMan Probe Mode of Action
18
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

19
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.

20
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.

21
LNA Base Structure
22
B and A Helices
23
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.

24
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.

25
Diagram of a Moleucular Beacon Probe
26
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.

27
Scorpion Primer Reaction Diagram
28
Technology Compatibility
29
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.

30
Fluorophore and Quencher Structures
  • Flourophore
  • Quencher

31
Fluorescence Principle
32
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

33
Proximal and FRET Quenching Diagram
  • Proximal
  • FRET

34
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.

35
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.

36
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

37
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

38
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

39
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.

40
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
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