Real Time PCR

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Real Time PCR

• Highly Accurate DNA Quantitation
• Assay for the Presence of PCR Inhibitors

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Strengths of Real-Time PCR

• Automation Friendly
• Not Labor Intensive
• Kits available for both Autosomal and Y DNA
  Quantification

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Drawbacks of Real-Time PCR

• Expensive
• Needs Validation

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Understanding Real-Time PCR

• Three Phases of PCR
• 1. Exponential- Growth Phase Reagents are
  available, kinetics drive the reaction forward,
  amplicons are doubling
• 2. Linear- Reagents are being consumed, reactions
  are slowing down
• 3. Plateau- Amplification has ceased. Gel
  detection

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Phases of PCR
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PCR of Three Replicates
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Exponential Phase with 5 Fold Dilutions
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Taq Man Assay

• 6- Carboxy Fluorescein dys at 5 End of Probe
• Non- Fluorescent Quencher (NFQ) at 3End of Probe
• FRET- Fluorescent Resonance Energy Transfer

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Taq Man Probe
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FRET Technology

• High energy 6-Fam dye is close to the NFQ the
  energy will transfer from high to low. As PCR
  products are formed the specific TaqMan probe
  will anneal to the amplicon. The Taq Polymerase
  will then cleave the Taq probe separating the NFQ
  and reporter dye. The fluorescence is no longer
  quenched and thus is proportional to the number
  of amplcions produced.

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TaqMan Assay Illustartion
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Internal Positive Control

• Found in both Autosomal and Y- specific Assays
• 10,000 copies of synthetic DNA template not found
  in nature
• PCR primer labeled with VIC
• Monitors PCR inhibition

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IPC Example
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Kit Components

• Human Male DNA Standard 200ng/ul dilited
  four-fold(50,12.5, 3.12, 0.78, 0.195, 0.049, and
  0.012 ng/ul)
• TaqMan 2X Universal PCR Master Mix
• Primer/Probe Mix

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Quantitation Standards
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Sequence Detection

• Halogen lamp excites the dyes
• Collection is at 500 and 660nm
• Lenses, filters and a dichroic mirror separartes
  the light by wavelengths onto a CCD
• Software uses algorithms to convert signals to
  quantification values

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Analysis

• Quantities are determined by monitoring cycle
  to-cycle changes in fluorescent during PCR. The
  fewer cycles it takes to reach detection, the
  greater the starting material

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Dyes

• 6FAM and VIC- Reporter Dyes
• ROX- Passive reference dye (Reference is found in
  equal concentrations in al samples). Rn
  (Normalized Reporter).
• Divide Reporter Signals by Rn to normalize them.

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Contributions of Each Dye
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Analysis Continued

• When Template Concentration reached 10-8 Molar,
  the exponential phase ends.
• CT Value- Calculated by software, point at which
  fluorescent signal crossed the threshold setting.

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Amplification Plot
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RT PCR vs Quantiblot
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RT PCR vs Quantiblot LCN Samples
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Female vs. Male DNA
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Male Crossover