Basic Principles of Real-Time PCR

Basic Principles of Real-Time PCR

Viraphong Lulitanond, Ph.D.
Research and Diagnostic Center for Emerging Infectious Diseases y , y Faculty of Medicine, Khon Kaen University viraphng@kku.ac.th

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Outlines for presentation

I. Why Real-Time PCR ? II. Real-Time PCR Detection Formats III. Quantitative Real-Time PCR IV. Application of Real-Time PCR

Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

I. Why Real-Time PCR ?

Why Real-time Amplification?

Question from Yes or No to How and/or How many? Qualitative PCR to Quantitative PCR
- Regulation of gene expression - Disease diagnosis - Therapeutic monitoring

Contamination issues Automation issues

Limitation of conventional PCR
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Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

Monitoring of PCR Reactions

Y = X(1+E)n

PCR and the Problem of Quantification

log-p log-phase analysis g y
endend-point analysis high concentration / g y high efficiency high concentration / low efficiency low concentration / high efficiency N : number of amplified molecules n : number of amplification cycles

Main problems of PCR: 1. Plateau effect 2. Efficiency of amplification

n
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Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

Competitive PCR

Endpoint Analysis by Gel Electrophoresis (1)

tPA: 109 108 107 106 105 104 103 102 101 H2O

500

250

comp.:

105

105

105

105

105

105

105

105

105

H2O

Detection of PCR products by Conventional PCR

Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

PCR ELISA

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Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

Area 1

Area 3

Area 2

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Limitations of Non Real-Time Quantification

Assumptions on reaction consistency and uniformity Narrow dynamic range Long optimisation and set up times Long run and analysis times g y High levels of inherent inaccuracy and variation
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Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

Development of Real-Time Analysis

First reported in 1992 by Higuchi et al. Used ethidium bromide to intercalate into double stranded (ds) DNA and a thermal cycler modified with a cooled charged coupled device (CCD camera) attached. PCR cycle = dsDNA = dye = fluorescence

Later changed to SYBR Green I as this has a much higher affinity for dsDNA rather than ssDNA compared to ethidium bromide.
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Viraphong Lulitanond, Ph.D.

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Realtime PCR
Schematic of LightCycler

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Viraphong Lulitanond, Ph.D.

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II. Real-time PCR Detection Formats

SYBR Green I

Hybridization Probe Format

Hydrolysis Probe Format

Elongation Phase

Annealing Phase

Elongation Phase
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Viraphong Lulitanond, Ph.D.

Basic Principles of Real-Time PCR

SYBR Green l Format Overview

D Denaturation: D bl strand t ti Double t d DNA to Single strand DNA by heat Primer Annealing: SYBR Green I start binding to minor grove of DNA strand Elongation : SYBR Green I has maximum Fluorescent signal can be monitored
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Monitoring of PCR with the LightCycler using SYBR Green I

H2O 1E+1 1E+2 1E+3 1E+4 1E+5 1E+6 1E+7 1E+8 1E+9

Fluorescence -d (F1) / dT

Fluorescence (F1)

Cycle Number

Temperature C

Template: Plasmid; Target: TNF; Detection Format: SYBR Green I

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Viraphong Lulitanond, Ph.D.

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Basic Principles of Real-Time PCR

Hybridization Probes Format

Fluorescein LC Red

transfer excite emission

Denaturation: Double Denaturation: strand DNA to Single strand DNA by heat. No FRET Probe hybridization : two oligo probe hybridize to DNA strand. Fluorescent signal can be monitored Elongation : Strand displacement by primer elongation

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Fluorescence Resonance Energy Transfer (FRET)

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Basic Principles of Real-Time PCR

Hybridization Probes: A Model Approach

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Hydrolysis Probe Format Overview

reporter
quencher

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Basic Principles of Real-Time PCR

III Quantitative Real-Time PCR III.

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Types of Quantitative Real-Time PCR

1. Absolute quantification 2. Relative quantification

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Basic Principles of Real-Time PCR

Absolute Quantification

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Can we use absolute quantitative real-time PCR for assessment of c-erb/Her2/neu gene duplication in breast tissues ?

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Basic Principles of Real-Time PCR

Relative Quantification

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IV. Applications of Real-Time PCR

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Basic Principles of Real-Time PCR

Derivation of Melting Curves Generates Melting Peaks

Differentiation of Multiplex PCR Products

Temperature C
Tumor Antigen GAPDH Mixed Templates

F Fluorescence -d (F1) / dT

Fluorescence (F1)

Temperature C

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Mutation Detection: Probe Design

perfect match

mismatch

Anchor Probe

Mutation Probe
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Tm of Mutation Probe approx. 5 C lower than Tm of Anchor Probe

Viraphong Lulitanond, Ph.D.

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Basic Principles of Real-Time PCR

Mutation Analysis with Hybridization Probes

Mismatch

Perfect match
Temperature

low

medium

high

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Mutation Analysis with Hybridization Probes

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Basic Principles of Real-Time PCR

Genotyping by Melting Peaks

Single Point Mutation (Factor V)

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Advantages of Real-Time Amplification over Conventional Methods

Sensitivity Speed Quantification and wide dynamic range Multiplex assay Mutation analysis Minimizing contamination issues Safety of data management Automation
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Basic Principles of Real-Time PCR

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