11

Actinobacteria as a Source of Novel Natural Products: Isolation, Molecular Characterization and Phylogenetic Analysis

Khartoum, March 28th-April 1st

Real Time PCRas a rapid

diagnostic tool

Dr. Mogahid M. Elhassan

SUST

Real-Time PCRReal-Time PCR

1.1.Why Real-time PCR? Advantages and Why Real-time PCR? Advantages and DisadvantagesDisadvantages

2.2.Theory of Real-time PCRTheory of Real-time PCR3.3.Types of Real-time PCR QuantificationTypes of Real-time PCR Quantification4.4.Choosing Housekeeping Gene for Choosing Housekeeping Gene for

NormalizationNormalization

Disadvantage of traditional PCRDisadvantage of traditional PCR

* Low sensitivity* Short dynamic range* Low resolution* Non-automated* Size-based discrimination only* Results are not expressed as numbers* Ethidium bromide staining is not very

quantitative

1. Why Real-time PCR ?

Advantages of real-time PCR

amplification can be monitored real-time amplification can be monitored real-time wider dynamic range of up to 1010-fold wider dynamic range of up to 1010-fold no post-PCR processing of productsno post-PCR processing of products ((No gel-based analysis at the end of the No gel-based analysis at the end of the

PCR reactionPCR reaction)) ultra-rapid cycling (30 minutes to 2 ultra-rapid cycling (30 minutes to 2

hours)hours) highly sequence-specifichighly sequence-specific

1. Why Real-time PCR ?

1.Requires expensive equipments and reagents

2.Due to its extremely high sensitivity, you may get high deviations of the same experiment, thus, the use of internal control genes is a recommended (in gene expression experiments)

Disadvantages of real-time PCRDisadvantages of real-time PCR

1. Why Real-time PCR ?

2.2. Theory of real-time Theory of real-time PCRPCR

Q-PCRQ-PCR Definition: Real-time Definition: Real-time

monitoring of the amplification monitoring of the amplification reaction.reaction.

Purpose: To estimate the initial Purpose: To estimate the initial quantity of specific template quantity of specific template DNA.DNA.

2- Theory of Real-time PCR ?

The QPCR ApproachThe QPCR Approach

ChemistryChemistry

� Use fluorescent dyes and probesUse fluorescent dyes and probes� Establish a linear correlation between PCR Establish a linear correlation between PCR

product and fluorescence intensityproduct and fluorescence intensity

DetectionDetection� Fluorescence detection to monitor Fluorescence detection to monitor

amplification in real timeamplification in real time

AnalysisAnalysis� Software for analysis and estimation of Software for analysis and estimation of

template concentrationtemplate concentration

2- Theory of Real-time PCR ?

10 X NH4 Buffer 5.0 µldNTP mix (12.5 mM) each 0.8 µlForward primer (20 µM) 1.0 µlReverse primer (20 µM) 1.0 µlMgCl2 3.0 µlSterile Milli-Q water 38.0 µlTaq polymerase 0.5 µl

10 X NH4 Buffer 5.0 µldNTP mix (12.5 mM) each 0.8 µlForward primer (20 µM) 1.0 µlReverse primer (20 µM) 1.0 µlMgCl2 3.0 µlSterile Milli-Q water 37.0 µlTaq polymerase 0.5 µlSybrGreen (50x) 1.0 µl

Reaction contents

ChemistryChemistry

2- Theory of Real-time PCR ?

How to measure the PCR How to measure the PCR productproduct

DirectlyDirectly• Sybr green Sybr green • Quality of primers criticalQuality of primers critical

IndirectlyIndirectly• In addition to primers, add a In addition to primers, add a

fluorescently labeled hybridization probefluorescently labeled hybridization probe

1111

2- Theory of Real-time PCR ?

ChemistriesChemistries

TaqManTaqMan®®

Molecular Molecular BeaconsBeacons

ScorpionsScorpionsTMTM AmplifluorAmplifluor®®

probes probes SYBR Green ISYBR Green ITMTM

Others...Others...

2- Theory of Real-time PCR ?

ChemistryChemistry

2- Theory of Real-time PCR ?

Fluorescence DetectionFluorescence Detection

LightLight LightExcitation Emission

2- Theory of Real-time PCR ?

REAL TIME REAL TIME PCRPCR

USING SYBR USING SYBR GREENGREEN

2- Theory of Real-time PCR ?

Sybr green is a dye which binds to Sybr green is a dye which binds to double stranded DNA but not to double stranded DNA but not to single-stranded DNA and is single-stranded DNA and is frequently used to monitor the frequently used to monitor the synthesis of DNA during real-time synthesis of DNA during real-time PCR reactions. When it is bound to PCR reactions. When it is bound to double stranded DNA it fluoresces double stranded DNA it fluoresces very brightly (much more brightly very brightly (much more brightly than ethidium bromide doesthan ethidium bromide does

2- Theory of Real-time PCR ?

SYBR Green AssaySYBR Green Assay

SYBR Green

SYBR GreenSYBR GreenSYBR GreenSYBR GreenSYBR Green

SYBR Green (high fluorescent conformation)

2- Theory of Real-time PCR ?

The TaqMan probe principle relies on the 5The TaqMan probe principle relies on the 5´–3´ nuclease activity of Taq polymerase ´–3´ nuclease activity of Taq polymerase to cleave a dual-labelled probe during to cleave a dual-labelled probe during hybridization to the complementary target hybridization to the complementary target sequence and fluorophore-based sequence and fluorophore-based detection. detection.

TaqMan probes consist of a fluorophore TaqMan probes consist of a fluorophore (Reporter) attached to the 5’-end of the (Reporter) attached to the 5’-end of the oligonucleotide probe and a quencher at oligonucleotide probe and a quencher at the 3’-end the 3’-end

2- Theory of Real-time PCR ?

Q RT PCR Using TaqMan

The quencher molecule quenches the The quencher molecule quenches the fluorescence emitted by the reporter fluorescence emitted by the reporter when excited by the cycler’s light when excited by the cycler’s light source via FRET (Fluorescence source via FRET (Fluorescence Resonance Energy Transfer).Resonance Energy Transfer).

As long as the reporter and the As long as the reporter and the quencher are in proximity, quenching quencher are in proximity, quenching inhibits any fluorescence signals inhibits any fluorescence signals

2- Theory of Real-time PCR ?

TaqMan ChemistryTaqMan Chemistry

R Q

2- Theory of Real-time PCR ?

TaqMan ChemistryTaqMan Chemistry

QRRRRRR

R

2- Theory of Real-time PCR ?

TaqMan ChemistryTaqMan Chemistry

Q

R

2- Theory of Real-time PCR ?

TaqMan ChemistryTaqMan Chemistry

Q

RR

2- Theory of Real-time PCR ?

Molecular BeaconsMolecular Beacons

QRR Q

R Q

R QR

Q QRQR

2- Theory of Real-time PCR ?

SCORPIONSSCORPIONS

Q

R

2- Theory of Real-time PCR ?

SCORPIONSSCORPIONS

Q

R

Q

R

Q

R

Q

R

Q

RQ RQ R

QR

QR

QRR

2- Theory of Real-time PCR ?

TaqmanTaqman BeaconBeacon

ScorpionScorpion

Comparison of Probe Chemistries Comparison of Probe Chemistries

2- Theory of Real-time PCR ?

Published References for real-time PCR Fluorescent Chemistries

0

20

40

60

80

100

120

140

160

180

1994 1995 1996 1997 1998 1999 2000 2001

Year

No

. of

refe

ren

ces

Taqman

SYBR Green

molecular beacons

scorpions

2- Theory of Real-time PCR ?

www.biorad.com

2a. excitation filters

2b. emission filters

1. halogen tungsten lamp

4. sample plate

3. intensifier5. detector 350,000 pixels

Optical Detection System of Real-Optical Detection System of Real-Time PCRTime PCR

2- Theory of Real-time PCR ?

Quantitative PCRQuantitative PCR[D

NA

]

Cycle #

Limit of detection

Ct

Threshold

2- Theory of Real-time PCR ?

Linear ground phase:•PCR is just began•Fluorescence emission at each cycle has not yet risen above background•Baseline fluorescence is calculated at this time

CT - threshold cycle:•the first significant increase in the amount of PCR product correlates to the initial amount of target template •CT represents the starting copy no. in the original template

Early exponential phase:•PCR is just began

•The amount of fluorescence has reached a threshold where it is significantly higher than background (usually 10 times the standard

deviation of the baseline)

PCR can be broken into 4 major phases

2. Theory of Real-time PCR2. Theory of Real-time PCR

2- Theory of Real-time PCR ?

3434

Types of Real Time PCR Quantification

3535

STANDARD CURVE METHOD

3. Types of Real-time PCR Quantification3. Types of Real-time PCR Quantification

3636

3. Types of Real-time PCR Quantification3. Types of Real-time PCR Quantification

3737

SERIES OF 10-FOLD DILUTIONS

3838SERIES OF 10-FOLD DILUTIONS

threshold

Ct

3. Types of Real-time PCR Quantification3. Types of Real-time PCR Quantification

3939

Dilut ion curve reference gene

‘copy number’ reference gene experimental

‘copy number’ reference gene control

3. Types of Real-time PCR Quantification3. Types of Real-time PCR Quantification

4040

∆∆Ct EFFICIENCY METHOD

APPROXIMATION METHOD

4141

IL1-b con

IL1-b vit

RPLP0 vit

RPLP0 con

4242

4343

IL1-b vit

RPLP0 vit

IL1-b con

RPLP0 con

av =19.80

av =19.93

av =18.03

av =29.63

∆ Ct = 9.70

∆ Ct = -1.7

∆ Ct = target - ref

∆ Ct = target - ref

Difference = ∆Ct-∆Ct= ∆∆Ct = 9.70-(-1.7)= 11.40

control

experiment

Standards Standards same copy number in all cellssame copy number in all cells expressed in all cellsexpressed in all cells no pseudogeneno pseudogene no alternate splicing in target PCR no alternate splicing in target PCR

region you want to amplify.region you want to amplify.

4444

4- Choosing Housekeeping Gene for Normalization4- Choosing Housekeeping Gene for Normalization

Standards Standards Commonly used standardsCommonly used standards

• Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase mRNAmRNA

• Beta-actin mRNABeta-actin mRNA• MHC I (major histocompatability complex I) MHC I (major histocompatability complex I)

mRNAmRNA• Cyclophilin mRNACyclophilin mRNA• mRNAs for certain ribosomal proteinsmRNAs for certain ribosomal proteins

E.g. RPLP0E.g. RPLP0 (ribosomal protein, large, P0; also (ribosomal protein, large, P0; also known as 36B4, P0, L10E, RPPO, PRLP0, 60S known as 36B4, P0, L10E, RPPO, PRLP0, 60S acidic ribosomal protein P0, ribosomal protein acidic ribosomal protein P0, ribosomal protein L10, Arbp or acidic ribosomal phosphoprotein L10, Arbp or acidic ribosomal phosphoprotein P0)P0)

• 28S or 18S rRNA28S or 18S rRNA4545

4- Choosing Housekeeping Gene for Normalization4- Choosing Housekeeping Gene for Normalization

Standards Standards

The perfect standard does not The perfect standard does not existexist

4646

4- Choosing Housekeeping Gene for Normalization4- Choosing Housekeeping Gene for Normalization

Applications of Q RT PCRApplications of Q RT PCR Gene expression (and microarray Gene expression (and microarray

validation).validation). DNA target quantification (nuclear, DNA target quantification (nuclear,

mitochondrial, residual DNA in protein preps mitochondrial, residual DNA in protein preps (QC)).(QC)).

SNP detection, Allele discrimination, SNP detection, Allele discrimination, Genotyping, HaplotypingGenotyping, Haplotyping

DNA Methylation, Apoptosis DNA Methylation, Apoptosis Viral load assays, pathogen & GMO Viral load assays, pathogen & GMO

detection.detection. Clinical Diagnostics (Cancer, Therapy Clinical Diagnostics (Cancer, Therapy

Response)Response)