Real-Time PCR Applications -Presentation by Nasr Sinjilawi

8/6/2019 Real-Time PCR Applications -Presentation by Nasr Sinjilawi

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Principles and Applications of Principles and Applications of Real-Time PCR inReal-Time PCR in

Molecular Diagnosis;Molecular Diagnosis;

Molecular InfectiousMolecular Infectious

&&

Molecular GeneticsMolecular Genetics..Prepared byPrepared by;;

Nasr A SinjilawiNasr A Sinjilawi

Technical Supervisor; Molecular Diagnostics Unit-KKUH Technical Supervisor; Molecular Diagnostics Unit-KKUH



What to knowWhat to know

??

--Overview of Basic PCR TechniqueOverview of Basic PCR Technique

-Comparison between Basic PCRComparison between Basic PCRand Real-Time PCRand Real-Time PCR

-

Principles of Real Time PCRPrinciples of Real Time PCR

-Some Applications of Real-TimeSome Applications of Real-Time

PCR in Medical FieldPCR in Medical Field



Overview of PCROverview of PCR

1. Temperature Cycling

Denaturation 94°

Annealing 55°Extension 72°

2. Every Cycle DNA

between primers is

duplicated



PCR AmplificationPCR Amplification



Exponential AmplificationExponential Amplification

30 cycles --- 2 Trillion copies in theory



PCR DemoPCR Demo



Principle of PCR



Disadvantages of basic PCR

What is Wrong with Agarose Gels?

Poor precision

Low specificity

Size-based discrimination only

Low sensitivity/Resolution

Short dynamic range (< 2 logs)

Possibility of human errors

Cross-contamination

Results are not expressed as numbers

Ethidium bromide staining is not very quantitative



What to know aboutWhat to know about

Real Time PCRReal Time PCR??

What is the basic principle of Real-TimeWhat is the basic principle of Real-TimePCRPCR??

What are the types of detection formatsWhat are the types of detection formats??

What are the types and applications of Real-What are the types and applications of Real-

Time PCR Time PCR??



What is the principle of What is the principle of

Real-Time PCRReal-Time PCR??

--Real time PCR is a technique used to monitor theReal time PCR is a technique used to monitor theprogress of a PCR reaction in real time. Atprogress of a PCR reaction in real time. At

the same time, a relatively small amount of PCRthe same time, a relatively small amount of PCRproduct (DNA, cDNA or RNA) can be quantified.product (DNA, cDNA or RNA) can be quantified.Real Time PCR is based on the detection of theReal Time PCR is based on the detection of the

fluorescence produced by afluorescence produced by a reporter moleculereporter molecule which increases, as the reaction proceedswhich increases, as the reaction proceeds..

-Real Time PCRReal Time PCR is based on the detection of theis based on the detection of thefluorescence produced by a reporter moleculefluorescence produced by a reporter moleculewhich increases, as the reaction proceeds. Thiswhich increases, as the reaction proceeds. This

occurs due to the accumulation of the PCR productoccurs due to the accumulation of the PCR productwith each cycle of amplificationwith each cycle of amplification..---fluorescent reporter moleculesfluorescent reporter molecules include dyes thatinclude dyes that

bind to the double-stranded DNA (i.e. SYBR® Greenbind to the double-stranded DNA (i.e. SYBR® Green) or sequence specific probes (i.e. FRET probes,) or sequence specific probes (i.e. FRET probes,

TaqMan® Probes, or Molecular Beacons TaqMan® Probes, or Molecular Beacons(.(.



Diversity of fluorescence dyes

Excitation/Emission wavelength

Position Excitation Emission Dye

1 470-490 520-530 Fluorescein, FAM, SYBR®Green

2 520 550-560 JOE, TET, VIC

3 550 580 TAMRA, CY3

4 580 610-640 Texas Red, ROX, RED610

5 640 670-705 CY5, RED 670



Work flow of Real-time PCRWork flow of Real-time PCR

Uni-directionl FlowUni-directionl Flow

((

1- Extraction of Pure Nucleic acids1- Extraction of Pure Nucleic acids

(DNA,RNA,or both DNA/RNA)(DNA,RNA,or both DNA/RNA)

2- Preparing of Master Mix Reagents2- Preparing of Master Mix Reagents((containing the detection reagentscontaining the detection reagents))

3- Run on Real-time PCR Machine3- Run on Real-time PCR Machine

4- Analyze the Results of PCR4- Analyze the Results of PCRproductsproducts

(time; 2-3 hours)(time; 2-3 hours)



Primer & ProbePrimer & Probe

Primer Primer Short Often < 50 nt( oligonucleotide sequence of DNAShort Often < 50 nt( oligonucleotide sequence of DNA

Complementary to the beginning and the end of the targetComplementary to the beginning and the end of the target

DNA sequenceDNA sequence

Needed to initiate the synthesis of new DNA in a PCRNeeded to initiate the synthesis of new DNA in a PCR

reactionreaction Involved inInvolved in AMPLIFICATIONAMPLIFICATION

ProbeProbe

A single-stranded DNA with a specific base sequenceA single-stranded DNA with a specific base sequence

Labeled with fluorescence dyes TaqMan probeLabeled with fluorescence dyes TaqMan probe

))

Used to detect the complementary base sequence of Used to detect the complementary base sequence of

target DNA/RNA by hybridizationtarget DNA/RNA by hybridization

Involved inInvolved in DETECTIONDETECTION

Reporter dye / Quencher dyeReporter dye / Quencher dye

Primer

Probe



10 pg

1 pg

1 pg ~ 500 org.

1 ng

100 pg

10 ng

100 ng

Real-Time PCR; On timeReal-Time PCR; On time

amplification and detectionamplification and detection



Threshold: the fluorescence measurement at which product can be

distinguished from background. Threshold should be set in the region

associated with an exponential growth of PCR product

Ct (Threshold cycle): The cycle number at which the fluorescence generated

within a reaction crosses the threshold. It is inversely correlated to the log

of he initial copy number

Real-Time PCR

Baseline

ThresholdCt

Amplification plot

cycle

F l u

o r e s c

e n c

e



Fluorescent detectorsFluorescent detectors

--None-specific FormatNone-specific Format;;

e.g.; SYBR Greene.g.; SYBR Green((---Specific Detection FormatSpecific Detection Format;;

- a- Hybridization probes; (FRET-probesa- Hybridization probes; (FRET-probes((b- Hydrolysis probes; (5’-Exonuclease activityb- Hydrolysis probes; (5’-Exonuclease activity((

c- Molecular Beacons; (hairpin activityc- Molecular Beacons; (hairpin activity((

d- Scorpion probesd- Scorpion probes..



--Binds to double-stranded DNABinds to double-stranded DNA

--Fluorescence increases 100X upon bindingFluorescence increases 100X upon binding

double-stranded DNAdouble-stranded DNA

--Cannot discriminate between desired versusCannot discriminate between desired versus

undesired productsundesired products

--Cheapest method for PCRCheapest method for PCR

--Look for increases in fluorescence withLook for increases in fluorescence withincreased productincreased product

SYBR green DyeSYBR green Dye



SYBR Green I formatSYBR Green I format

overviewoverview



Intercalating method

SYBR Green dye

1. Advantages:

- Cheap, easy to use

- Does not inhibit the reaction of amplification

- Does not require any fluorescent probe- Does not require any particular expertise for the design of the probes

- Is not affected by mutations in the target DNA

2. Disadvantages:

- Impossible to make sure of specificity of amplicons

- Bad pairing can lead to positive forgeries or an over-estimate of the

quantification

- Still unspecified mutagen capacity



Hybridization probeHybridization probe

FormatFormat

Annealing phase methodAnnealing phase method Uses two probesUses two probes

--One regular probe is labeled with aOne regular probe is labeled with a Donor fluorophore (DDonor fluorophore (D((

-- The second probe also has an The second probe also has an Acceptor Fluorophore (AAcceptor Fluorophore (A((

--Annealing of first and second probes allows FRET,Annealing of first and second probes allows FRET,

during the annealing stageduring the annealing stage

--Look for increase in fluorescenceLook for increase in fluorescence..



Fluorescence ResonanceFluorescence Resonance

Energy Transfer FRETEnergy Transfer FRET((

24



1( FRET method designed two

specifically probe. It labeled with

different dyes, such as at the 5’

end of donor probe and at the 3’

end of acceptor probe.

2( At close proximity, the donor dye

is excited by the light source and

the energy is transferred the

acceptor dye. Subsequently,

fluorescent light is emitted at a

different wavelength.

2( Annealing

1( Denaturation

3( Extension

D A

Taq

D A

D A

Energy

transfer

Hybridization Probe (FRET)

FRET Probe



Hybridization probe formatHybridization probe format

overview FREToverview FRET((



Hydrolysis Probe Format;Hydrolysis Probe Format;

TaqMan; 5’-Exonulase activityTaqMan; 5’-Exonulase activity

--Uses one probeUses one probe

--Contains two fluorescent compounds: aContains two fluorescent compounds: a Reporter (R)Reporter (R) and aand a Quencher (QQuencher (Q((

--Probe is degraded by the exonuclease activity of Probe is degraded by the exonuclease activity of TaqTaq polymerase polymerase

Reduces the FRET (fluorescence resonance energyReduces the FRET (fluorescence resonance energytransfer) of signal from thetransfer) of signal from the RR to theto the QQ

--Releasing of Reporter fluorophore will emit theReleasing of Reporter fluorophore will emit thedetectable Light. ( in positive casesdetectable Light. ( in positive cases((



1( Fluorescent reporter dye at the 5’

end is quenched by fluorescent

quencher dye at the 3’ end.

2( When amplification occurs the

TaqMan probe is degraded due

to the 5'-->3' exonuclease activity

of Taq DNA polymerase, thereby

separating the quencher from thereporter during extension.

3( The TaqMan assay accumulates

a fluorescence signal.

2( Annealing

1( Denaturation

3( Extension

QR

R Q

Taq

RQ

R Q

TaqMan® probe

TaqMan Probe



TaqMan® probe

TaqMan Probe

1. Advantages:

- Increased specificity

- Better capacity of multiplexing

2. Disadvantages:

- Little expensive (dual-labeled probe)

- Less effective and less flexible compared to other techniques in the

real-time detection of specific mutation- Require the design of probes



Molecular Beacon; HairpinMolecular Beacon; Hairpin

looploop

--probe is quenched when in hairpin formprobe is quenched when in hairpin form..

--Extended form has brighter signalExtended form has brighter signal..

--Incorporated probe as the brightest signalIncorporated probe as the brightest signal..

--Look for increased fluorescence withLook for increased fluorescence with

increased productincreased product..



1( A molecular beacon begins as a

stem-and-loop structure. The

sequences at the ends of the

probe match and bind, creating

the stem

2( When the probe binds to a single-

stranded DNA template, thestructure unfolds, separating the

quencher from the dye and

allowing fluorescence.

2( Annealing

1( Denaturation

3( Extension

QF

QTaq F

QF

Molecular Beacon

Molecular Beacon Probe



Molecular Beacon

Molecular Beacon Probe

1. Advantages:

- Increased specificity

- High flexibility for probe design- As the probes are not hydrolyzed, they are used at each cycle

2. Disadvantages:

- Little expensive (dual-labeled probe)

- Less effective and less flexible compared to other techniques in the

real-time detection of specific mutation

- Require the design of probes



Summary

Real-time PCR vs. Conventional PCR

Real-Time PCRReal-Time PCR Basic-PCRBasic-PCR

SensitivitySensitivity HighHigh LowLow

SpecificitySpecificity HighHigh

--use specific probesuse specific probes

LowLow

--only size discriminationonly size discrimination

QuantitativeQuantitativeresultsresults

Yes Yes--Specific fluorescenceSpecific fluorescence

NoNo--EtBr stainingEtBr staining

Detection methodDetection method Probe-specific FluorescenceProbe-specific Fluorescence Agarose gelAgarose gelElectrophoresisElectrophoresis

Detection rangeDetection range

Wide rangeWide range

Short range (<2 logShort range (<2 log((Reaction timeReaction time 11hr hr 3-53-5hr hr

Post-PCR stepsPost-PCR steps NoNo Agarose gel electrophoresisAgarose gel electrophoresis

Cross-Cross-contaminationcontamination

NoNo--Closed systemClosed system

--Single stepSingle step

Yes Yes--Open systemOpen system

--Multiple stepsMultiple steps



Real-Time PCR VS Basic PCRReal-Time PCR VS Basic PCR



PCR Agarose gel electrophoresis

The final product UV visualization

3-4 hours

The LightCycler SystemThe LightCycler System



Real-Time PCR instrumentsReal-Time PCR instruments

Carousel-basedsystems; 32 capillaries

Plate-based system; 96-wells(20-100 µl capacity) or 384

wells (5-20 µl capacity)

LightCyclerV.2

LightCyclerV.1 ,1.5

LightCycler 480

20µl Capacity20-100µl

capacity



LightCycler V.1,1.5 SystemLightCycler V.1,1.5 System



LightCycler V.2 SystemLightCycler V.2 System

6channels;530,560,610,640,670,and 705 nm

LightCycler V.2;wide applications



Real Time PCR applications inReal Time PCR applications in

Medical FieldMedical Field

11--Qualitative applicationsQualitative applications

A- detection (e.g., HSV1/2,H1N1, …etcA- detection (e.g., HSV1/2,H1N1, …etc((

B- Genotyping (HSV1/2, …etcB- Genotyping (HSV1/2, …etc((

C- Detection of Mutations (F-II,F-V,MTHFR,C- Detection of Mutations (F-II,F-V,MTHFR,IL-6 promoter, JAK2 V617F, …etcIL-6 promoter, JAK2 V617F, …etc((

22--QuantitativeQuantitative

A- absolute Quantitative Parvovirus B19,A- absolute Quantitative Parvovirus B19,

…etc…etc((

B- Relative Quantitative ( HCV monitor ,HBVB- Relative Quantitative ( HCV monitor ,HBVmonitor, HIV monitor, …etcmonitor, HIV monitor, …etc((



Detection of Viral agentsDetection of Viral agents

e.g. HSV1/2e.g. HSV1/2 DNA virus DNA virus((

CtCt

Denaturation Amplification MeltingCurve

Coolingat 40 0C

Positive CSFsample

Negative control

Positive control



Genotyping of Viral agentsGenotyping of Viral agents

e.g. HSV1/2e.g. HSV1/2 DNA virus DNA virus((

Data

Analysis;

Melting Curve

Analysis

Hybridizationprobe format



HSV1/2 Detection and Genotyping; Data AnalysisHSV1/2 Detection and Genotyping; Data Analysis

HSV1; Tm= 68± 2.5 oC

HSV2; Tm= 54 ± 2.5oC

Internal Control (IC);60 ± 2.5 oC

54oC 68oC60oC

Hybridizationprobe format

D t ti f Vi l tD t ti f Vi l t



Detection of Viral agentsDetection of Viral agents

e.g. H1N1 Swine Flue.g. H1N1 Swine Flu((

InFA/H1N1positive

NegativeControl

InfApositive

ReverseTranscription

Amplification

Coolingat 40 0C



F-II Mutation DetectionF-II Mutation Detection

11

22 33

Continue



Mutation Detection probesMutation Detection probes



Mutation DetectionMutation Detection

F II t ti d t ti D t



F-II mutation detection, DataF-II mutation detection, Data

AnalysisAnalysis

Tm = 51 oC

Melting Peak Analysis;

No Any Sample ShowsPositive Homozygous

full mutation at LowerTem.

Positive Heterozygouscarrier

Negative HomozygousWild-type

H2O

Tm = 61o

C

Hybridization

probe format



F-V Mutation DetectionF-V Mutation Detection

11

2233

Continue



F-V mutation detection, Data AnalysisF-V mutation detection, Data Analysis

Tm = 57 oC Tm = 65 oC

NegativeHomozygousWild-type

PositiveHeterozygouscarrier

PositiveHomozygous

Mutant

Hybridization

probe format

Factor VFactor V Melting CurveMelting Curve((



Factor VFactor V Melting CurveMelting Curve((

F tF t



Diagnostic Molecular BiologyDiagnostic Molecular Biology

Unit/KKUH,1428Unit/KKUH,1428

Factor vFactor v Peak AreaPeak Area((

QuestionsQuestions??????



QuestionsQuestions??????

Sample No.1Sample No.1



Quantification



QuantificationStrategies

Absolute quantification - Used to quantitate unknown samples by interpolating their

quantity from a standard curve.

- The standard is a known DNA sample whose concentration is

known absolutely.

- The accuracy of the absolute quantification assay is entirelydependent on the accuracy of the standards.

Relative quantification

- Used to analyze changes in gene expression in a given sample

relative to another reference sample.

- A comparison within a sample (DNA or cDNA) is made with the

gene(s) of interest to that of an endogenous control gene.

- Quantification is done relative to the control gene.

Absolute Quantification



The Ct value correlates strongly with the starting copy number.It is linear with the log of starting DNA concentration.

Ct value


( e.g. parvovirus B19)

Sample 1Ct:14

Conc: 2,500copyThreshold




Relative QuantitativeRelative Quantitativee.g. ;HCV monitor, HBV monitor e.g. ;HCV monitor, HBV monitor



HCV monitor HCV monitor

Amplification plotAmplification plot

Target NotDetected

624054IU/ml



HBV monitor HBV monitor



HBV amplification plotHBV amplification plot

Target notdetected

1589058IU/ml

NP Genotyping



Single Nucleotide Polymorphism (SNP)

DNA sequence variations that occur when a single nucleotide (A, T,C, or G) in the genome sequence is altered

How many SNPs are there in humans today?

- Human mutation rate is ~ 2.5 x 108 mutation/site/generation- ~150 mutations/diploid genome/generation- 6.3 billion people in the world = 945,000,000,000 mutations in the

world today

The most common type of sequence difference between alleles

Provide a way to detect direct associations betweenallelic forms of genes and phenotypes

NP Genotyping

P Genotyping Real-Time PCR(



Allelic Discrimination Assays (Single Nucleotide Polymorphisms(

G

G

C C

T

T

A A

P Genotyping Real Time PCR(Hydrolysis probeformat

SNPSNP



SNPSNP

--Single Nucleotide PolymorphismsSingle Nucleotide Polymorphisms

–

--Allelic Discrimination AssaysAllelic Discrimination Assays

–

--Genomic locus where two or more alternative bases occur with appreciable frequencyGenomic locus where two or more alternative bases occur with appreciable frequency

P Genotyping Real-Time PCR(



P Genotyping Real Time PCR(

A

A

A

T

T

T

G

G

G

C

CC

530nm

570nm

530nm

530nm

570nm570nm

Mutant

Carrier

Wild-type

JAK2 V617F t ti d t tiJAK2 V617F mutation detection



JAK2 V617F mutation detectionJAK2 V617F mutation detection

JAK2 V617F mutationJAK2 V617F mutationResult RS-ratio FAM/VIC ratio FAM/VIC FAM 530 VIC 560 nm samples



NEG 0.628 0.599 0.598 4.833 8.078 1: 1304-776710

0.600 4.767 7.945 2: 1304-776710

NEG 0.628 0.598 0.598 4.860 8.133 3: 1330-641222

0.598 4.849 8.109 4: 1330-641222

NEG 0.628 0.595 0.595 4.723 7.938 5: 1431-673876

0.596 4.852 8.148 6: 1431-673876

POS 0.628 0.826 0.826 8.420 10.199 7: 1468-919815

0.826 8.585 10.388 8: 1468-919815

NEG 0.628 0.593 0.592 4.651 7.858 9: 1481-343610

0.594 4.619 7.770 10: 1481-343610

POS 0.628 0.938 0.937 9.027 9.633 11: 1525-402499

0.940 9.195 9.787 12: 1525-402499

POS 0.628 0.989 0.988 9.377 9.488 13: 1536-037263

0.990 9.047 9.136 14: 1536-037263

NEG 0.628 0.596 0.594 4.518 7.611 15: 1544-5819680.598 4.782 8.003 16: 1544-581968

NEG 0.628 0.593 0.593 4.793 8.081 17: 1656-254784

0.594 4.724 7.958 18: 1656-254784

NEG 0.628 0.589 0.589 4.891 8.307 19: 1658-020564

0.590 4.884 8.276 20: 1658-020564

POS 0.628 0.734 0.746 7.060 9.467 21: 1671-950406

0.721 6.696 9.282 22: 1671-950406

NEG 0.628 0.592 0.592 4.584 7.744 23: 1703-964800

0.592 4.753 8.034 24: 1703-964800

NEG 0.628 0.592 0.591 4.939 8.351 25: 1704-902480

0.592 4.956 8.373 26: 1704-902480

POS 0.628 1.075 1.073 9.931 9.252 27: PC

1.076 10.057 9.343 28: PC

NEG 0.628 0.583 0.582 4.961 8.518 29: NG

0.583 4.662 7.994 30: NG

RS 0.628 0.628 0.624 5.287 8.470 31: RS

ultiplex Analysis



ultiplex Analysis

Different dyes for each target (Example: FAM, TET, VIC and JOE)

Real-time detection of four differentretroviral DNAs in a multiplex format.

Each reaction contained four sets of PCR primers specific for unique HIV-1, HIV-2,HTLV-I, and HTLV-II nucleotidesequences and four molecular beacons,

each specific for one of the fouramplicons and labelled with a differentlycoloured fluorophore.

HIV-1: Fluorescein

HIV-2: Tetrachlorofluorescein

HTLV-1: Tetramethylrhodamine

HTLV-II: Rhodamine

Vet JA et al. PNAS (1999)

SummarySummary



SummarySummary- Real Time PCR is Quantitative andReal Time PCR is Quantitative and

Qualitative Technique ( has wide range of Qualitative Technique ( has wide range of applications)applications)

-Real Time PCR is Highly specific andReal Time PCR is Highly specific and

sensitive rather than basic PCRsensitive rather than basic PCR

-Low possibility of contamination (close tubeLow possibility of contamination (close tube

system)system)

- Less time consuming and less effortLess time consuming and less effort

(amplification and detection at the same(amplification and detection at the sameTime).Time).

- Expensive and need well trained people.Expensive and need well trained people.



Thank you Thank you

Real-Time PCR Applications -Presentation by Nasr Sinjilawi

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