Introduction To QPCR

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Introduction To Q-PCR
James Mellott Q-PCR Applications Scientist
Stratagene james.mellott_at_stratagene.com
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Q-PCR

• Definition Real-time monitoring of the
  amplification reaction.
• Purpose To estimate the initial quantity of
  specific template DNA.
• Advantages Sensitivity, reproducibility, dynamic
  range, throughput.

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The QPCR Approach

• Chemistry
• Use fluorescent dyes and probes
• Establish a linear correlation between PCR
  product and fluorescence intensity
• Detection
• Fluorescence detection to monitor amplification
  monitored in real time
• Analysis
• Software for analysis and estimation of template
  concentration

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Quantitative PCR
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Relative Quantitation
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Definition Ct value
Ct Fractional cycle number at which the
fluorescence intensity crosses the arbitrary
threshold line.
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Applications

• Gene expression (and microarray validation).
• DNA target quantification (nuclear,
  mitochondrial, residual DNA in protein preps
  (QC)).
• SNP detection, Allele discrimination, Genotyping,
  Haplotyping
• DNA Methylation, Apoptosis
• Viral load assays, pathogen GMO detection.
• Clinical Diagnostics (Cancer, Therapy Response)
• Static reads (96-well fluorimeter).

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Chemistry
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Chemistries

• TaqMan
• Molecular Beacons
• ScorpionsTM
• Amplifluor probes
• SYBR Green ITM
• Others...

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Fluorescence Detection
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SYBR Green Assay
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TaqMan Chemistry
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TaqMan Chemistry
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TaqMan Chemistry
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TaqMan Chemistry
Q
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Molecular Beacons
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SCORPIONS
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SCORPIONS
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Comparison of Probe-Based QPCR Chemistries
Scorpion
Beacon
TaqMan
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Comparison of Probe Chemistries
Taqman
Beacon
Scorpion
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