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Real-Time Quantitative PCR Basis

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Title: Real-Time Quantitative PCR Basis


1
Real-Time Quantitative PCRBasis
2
ABI Prism 7900HT real-time PCR instrument
3
Content
  • Principles of quantification
  • Methods of quantification
  • Applications

4
  • The polymerase chain reaction (PCR) has
    revolutionized the detection of DNA and RNA. As
    little as a single copy of a particular sequence
    can be specifically amplified and detected.
  • Theoretically, there is a quantitative
    relationship between amount of starting target
    sequence and amount of PCR product at any given
    cycle. In practice, though, it is a common
    experience for replicate reactions to yield
    different amounts of PCR product.
  • The development of real-time quantitative PCR has
    eliminated the variability traditionally
    associated with quantitative PCR, thus allowing
    the routine and reliable quantitation of PCR
    products.

5
PCR Reaction Phase
6
Quantification in Log Phase
  • Initial template ,more significant
  • Perfect reproducibility, less error
  • Constant amplification efficiency, linear
    standard curve

7
Several Basic Concepts
  • Threshold The low limit when PCR reaction signal
    goes into log phase
  • Ct value The cycle number of PCR reaction when
    the fluorescence signal reaches to threshold

8
Standard Curve
  • CT -k lgX0 b
  • There is a linear relationship between CT and
    lgX0 (X0 stands for amount of initial DNA)
  • Use standard sample which we have known its
    initial concentration to make standard curve. And
    then we can identify the initial concentration of
    unknown template through the standard curve when
    we obtain its CT value.

9
Methods of quantification
  • Dye method
  • SYBR Green I
  • Probe methods
  • TaqMan Probe and TaqMan MGB Probe
  • Molecular beacons
  • Dual-oligo FRET pairs

10
SYBR Green I
  • SYBR Green I dye is a highly specific
    double-stranded DNA binding dye. Its fluorescence
    increases when it bound to double-stranded DNA
    while disappears when DNA denatured.
  • There is a direct ratio between fluorescence
    signal and the amount of double-stranded DNA.

11
Advantages and Disadvantages about SYBR Green I
  • SYBR Green I assay chemistry will detect all
    double-stranded DNA, including non-specific
    reaction products.
  • The advantage of SYBR Green I assay chemistry
    is that no probe is required, thus reducing
    assay setup and running costs. And it can make
    dissociation curve analysis.

12
Fluorescent Resonant Energy Transfer
(FRET)
  • When the emission band of one fluorophore is
    overlapped with the absorption band of another,
    and simultaneously they are very near , then the
    energy will transfer from short wavelength (high
    energy) fluorophore to the longer wavelength
    (lower energy ) one. In other words, the
    fluorescence of short wavelength fluorophore is
    quenched by another.

13
TaqMan Probe
  • One probe, two primers.
  • One probe, two fluorophores one is reporter,
    another is quencher .
  • Hybridises with the target amplicon?
  • Is 3 terminally blocked (cannot be extended by
    the polymerase)?
  • Has two fluorescent dyes attached
  • 1.Reporter(R)
  • 2.Quencher(Q)

14
React Process
15
  • The production of one DNA strand will cut one
    probe into pieces simultaneously.
  • The disjunction of one probe will produce one
    fluorescent signal.
  • Signal intensity are proportional to the amount
    of DNA which is binded by probe.

16
Advantages about TaqMan chemistry
  • Noiseless data
  • Due to the second level of specificity
    provided by the probe.
  • Multiplex compatible
  • Each probe can be differently colored and
    thereby mixed with others.
  • Signal proportional to products
  • Signal related to amount of amplified product
    .
  • Unreversible reaction, signal will not quenched
  • Other probe chemistries use reversible
    hybridisation to generate signal.

17
TaqMan MGB Probe
  • Non background fluorescence
  • All probes will be short(13-20bp)
  • Tm enhancer MGB
  • Minor Groove Binder attached at 3' end of probe

18
SNP Discrimination (Allele Discrimination
)
19
Result
  • Homozygote
  • wild type FAM
  • mutant type VIC
  • Heterozygote
  • FAMVIC

20
Other Methods Based on Probe
  • Molecular beacons
  • Dual-oligo FRET pairs

21
Applications Primer and Probe Design Using
Primer Express Software
  • Primer Express software uses a set of default
    parameters to automatically select primer and
    probe sets.
  • Even though no probe is required for SYBR Green
    I dye detection, it is still a good idea to use
    Primer Express software to select a primer and
    probe set when designing a SYBR Green I assay.
    Although no probe will be used, the primers will
    meet all the required criteria and if, in the
    future, there is the need to convert the assay to
    TaqMan assay chemistry to obtain higher
    specificity, the probe can immediately be found
    in the original Primer Express software
    document.

22
Primer and Probes selection guidelines for
quantitative assays
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Applications
  • Absolute Quantification (AQ)
  • Absolute--produces data with quantity amounts
  • The absolute quantity of target gene is measured
  • needs a standard template whose concentration is
    known absolutely
  • The quantity of the standard template has to be
    measured precisely
  • unnecessary for most studies

25
Applications
  • Relative Quantification (RQ)
  • Relative--makes comparisons of quantity (no
    units)
  • relative standard curve or ??CT analysis
  • any stock DNA or RNA containing the target gene
    can be used to prepare relative standard curve
  • normalises for amount of sample added
  • needs endogenous control target
  • the most powerful and widely used method

26
Applications
  • Allelic Discrimination (AD)
  • Plus/Minus with IPC (/-)

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THANKS!
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