Real-Time Quantitative PCR Basis

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Real-Time Quantitative PCRBasis
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ABI Prism 7900HT real-time PCR instrument
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Content

• Principles of quantification
• Methods of quantification
• Applications

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• 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.

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PCR Reaction Phase
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Quantification in Log Phase

• Initial template ,more significant
• Perfect reproducibility, less error
• Constant amplification efficiency, linear
  standard curve

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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

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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.

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Methods of quantification

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

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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.

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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.

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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.

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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)

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React Process
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• 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.

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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
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• Unreversible reaction, signal will not quenched
• Other probe chemistries use reversible
  hybridisation to generate signal.

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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

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SNP Discrimination (Allele Discrimination
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Result

• Homozygote
• wild type FAM
• mutant type VIC
• Heterozygote
• FAMVIC

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Other Methods Based on Probe

• Molecular beacons
• Dual-oligo FRET pairs

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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.

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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

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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

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Applications

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

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