Principles and Applications of Real-Time PCR in Molecular Diagnosis; Molecular Infectious & Molecular Genetics

;Prepared by . Nasr A Sinjilawi Technical Supervisor; Molecular Diagnostics Unit-KKUH

?What to know
Overview of Basic PCR Technique-Comparison

between Basic PCR and Real-Time PCR of Real Time PCR

-Principles -Some

Applications of Real-Time PCR in Medical Field

Overview of PCR
1. Temperature Cycling Denaturation Annealing Extension 94 55 72

2. Every Cycle DNA between primers is duplicated

PCR Amplification

Exponential Amplification

30 cycles --- 2 Trillion copies in theory

PCR 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 about ?Real Time PCR

What is the basic principle of Real-Time ?PCR ?What are the types of detection formats What are the types and applications of Real?Time PCR

What is the principle of ?Real-Time PCR

Real time PCR is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule .which increases, as the reaction proceeds Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product .with each cycle of amplification fluorescent reporter molecules include dyes that - bind to the double-stranded DNA (i.e. SYBR Green ) or sequence specific probes (i.e. FRET probes, (.TaqMan Probes, or Molecular Beacons

Diversity of fluorescence dyes

Excitation/Emission wavelength
Position 1 2 3 4 5 Excitation Emission 470-490 520 550 580 640 Dye

520-530 Fluorescein, FAM, SYBRGreen 550-560 580 610-640 670-705 JOE, TET, VIC TAMRA, CY3 Texas Red, ROX, RED610 CY5, RED 670

Work flow of Real-time PCR ((Uni-directionl Flow 1- Extraction of Pure Nucleic acids (DNA,RNA,or both DNA/RNA) 2- Preparing of Master Mix Reagents (containing the detection reagents) 3- Run on Real-time PCR Machine 4- Analyze the Results of PCR products (time; 2-3 hours)

Primer & Probe

Primer
Short (Often < 50 nt( oligonucleotide sequence of DNA Complementary to the beginning and the end of the target DNA sequence Needed to initiate the synthesis of new DNA in a PCR reaction Involved in AMPLIFICATION
Probe Primer

Probe
A single-stranded DNA with a specific base sequence )Labeled with fluorescence dyes (TaqMan probe Used to detect the complementary base sequence of target DNA/RNA by hybridization Involved in DETECTION Reporter dye / Quencher dye

Real-Time PCR; On time amplification and detection

100 ng 10 ng 1 ng 100 pg

10 pg

1 pg

1 pg ~ 500 org.

Real-Time PCR
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

Amplification plot

Ct

Threshold

c necser ou F l

Baseline

cycle

Fluorescent detectors
;None-specific Format(e.g.; SYBR Green ( ;Specific Detection Format- ( a- Hybridization probes; (FRET-probes (b- Hydrolysis probes; (5-Exonuclease activity ( c- Molecular Beacons; (hairpin activity .d- Scorpion probes

SYBR green Dye

Binds to double-stranded DNAFluorescence increases 100X upon binding double-stranded DNA Cannot discriminate between desired versusundesired products Cheapest method for PCRLook for increases in fluorescence with increased product

SYBR Green I format overview

SYBR Green dye

Intercalating method 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 probe Format

Annealing phase method
Uses two probes
(One regular probe is labeled with a Donor fluorophore (D(The second probe also has an Acceptor Fluorophore (A-

Annealing of first and second probes allows FRET,during the annealing stage .Look for increase in fluorescence -

Fluorescence Resonance (Energy Transfer (FRET

24

FRET Probe
Hybridization Probe (FRET) 1( Denaturation
D A

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( Annealing
Taq D A

Energy transfer

2(

3( Extension
D A

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.

Hybridization probe format (overview (FRET

Hydrolysis Probe Format; TaqMan; 5-Exonulase activity

Uses one probeContains two fluorescent compounds: a Reporter (R) (and a Quencher (Q Probe is degraded by the exonuclease activity of Taqpolymerase Reduces the FRET (fluorescence resonance energy transfer) of signal from the R to the Q Releasing of Reporter fluorophore will emit the (detectable Light. ( in positive cases -

TaqMan Probe
TaqMan probe 1( Denaturation
R Q

1(

Fluorescent reporter dye at the 5 end is quenched by fluorescent quencher dye at the 3 end.

2( Annealing
Taq R Q

2(

3( Extension
R Q

When amplification occurs the TaqMan probe is degraded due to the 5'-->3' exonuclease activity of Taq DNA polymerase, thereby separating the quencher from the reporter during extension.

3(

The TaqMan assay accumulates a fluorescence signal.

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; Hairpin loop

.probe is quenched when in hairpin form.Extended form has brighter signal. Incorporated probe as the brightest signalLook for increased fluorescence with.increased product

Molecular Beacon Probe

Molecular Beacon 1( Denaturation
F Q

1(

2( Annealing
Taq F Q

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(

3( Extension
F Q

When the probe binds to a singlestranded DNA template, the structure unfolds, separating the quencher from the dye and allowing fluorescence.

Molecular Beacon Probe

Molecular Beacon 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 PCR
Sensitivity Specificity Quantitative results High High use specific probesYes Specific fluorescence-

Basic-PCR
Low Low only size discriminationNo EtBr stainingAgarose gel Electrophoresis (Short range (<2 log hr 3-5 Agarose gel electrophoresis Yes Open systemMultiple steps-

Detection method Probe-specific Fluorescence Detection range Reaction time Post-PCR steps Crosscontamination Wide range hr 1 No No Closed systemSingle step-

Real-Time PCR VS Basic PCR

PCR

Agarose gel electrophoresis

3-4 hours

The LightCycler System

The final product UV visualization

Real-Time PCR instruments

LightCycler V.2

LightCycler 480

LightCycler V.1 ,1.5 l Capacity 20 l 20-100 capacity

Carousel-based systems; 32 capillaries

Plate-based system; 96-wells (20-100 l capacity) or 384 )wells (5-20 l capacity

LightCycler V.1,1.5 System

LightCycler V.2 System

LightCycler V.2; wide applications

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

Real Time PCR applications in Medical Field Qualitative applications- 1 ( A- detection (e.g., HSV1/2,H1N1, etc (B- Genotyping (HSV1/2, etc C- Detection of Mutations (F-II,F-V,MTHFR, ( IL-6 promoter, JAK2 V617F, etc Quantitative- 2 A- absolute Quantitative Parvovirus B19, ( etc B- Relative Quantitative ( HCV monitor ,HBV ( monitor, HIV monitor, etc

Detection of Viral agents (e.g. HSV1/2 ( DNA virus

Denaturation

Amplification

Melting Curve

Cooling at 40 0C

Positive control

Positive CSF sample

Ct

Ct

Negative control

Genotyping of Viral agents (e.g. HSV1/2 ( DNA virus

Data ;Analysis
Melting Curve Analysis

Hybridization probe format

HSV1/2 Detection and Genotyping; Data Analysis

Hybridization probe format

HSV1; Tm= 68 2.5 oC HSV2; Tm= 54 2.5 o C Internal Control (IC); 60 2.5 oC

C 54

C 60

C 68

Detection of Viral agents (e.g. H1N1 ( Swine Flu

Amplification
Reverse Transcription

Cooling at 40 0C

InFA/H1N1 positive InfA positive

Negative Control

F-II Mutation Detection

1

Continue

Mutation Detection probes

Mutation Detection

F-II mutation detection, Data Analysis Hybridization

probe format
Positive Heterozygous carrier Negative Homozygous Wild-type

H2O

;Melting Peak Analysis No Any Sample Shows Positive Homozygous full mutation at Lower .Temp
Tm = 61 oC

Tm = 51 oC

F-V Mutation Detection

1

Continue

F-V mutation detection, Data Analysis

Negative Homozygous Wild-type Positive Homozygous Mutant Positive Heterozygous carrier

Hybridization probe format

Tm = 57 oC

Tm = 65 oC

Factor V (Melting Curve

Factor v (Peak Area

Diagnostic Molecular Biology Unit/KKUH,1428

???Questions

Sample No.1

Sample No.2

Sample No.3

Quantification Strategies
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 entirely dependent 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
Absolute Quantification ( e.g. parvovirus B19)

Ct value

Threshold

Sample 1 Ct:14 Conc: 2,500copy

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

Relative Quantitative
e.g. ;HCV monitor, HBV monitor

HCV monitor Amplification plot

IU/ml 624054

Target Not Detected

HBV monitor

HBV amplification plot

IU/ml 1589058

Target not detected

SNP 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 between allelic forms of genes and phenotypes

NP Genotyping (Real-Time PCR(

Hydrolysis probe format

Allelic Discrimination Assays (Single Nucleotide Polymorphisms(

T A G A

G C

T C

SNP
Single Nucleotide PolymorphismsAllelic Discrimination AssaysGenomic locus where two or more alternative bases occur with appreciable frequency

NP Genotyping (Real-Time PCR(

G C G C

T A T A

T A G C

nm 530

Mutant
nm 530 nm 570 nm 530 nm 570

Wild-type
nm 570

Carrier

JAK2 V617F mutation detection

JAK2 V617F mutation

Result NEG NEG NEG POS NEG POS POS NEG NEG NEG POS NEG NEG POS NEG RS RS-ratio 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 0.628 FAM/VIC ratio 0.599 0.598 0.595 0.826 0.593 0.938 0.989 0.596 0.593 0.589 0.734 0.592 0.592 1.075 0.583 0.628 FAM/VIC 0.598 0.600 0.598 0.598 0.595 0.596 0.826 0.826 0.592 0.594 0.937 0.940 0.988 0.990 0.594 0.598 0.593 0.594 0.589 0.590 0.746 0.721 0.592 0.592 0.591 0.592 1.073 1.076 0.582 0.583 0.624 FAM 530 4.833 4.767 4.860 4.849 4.723 4.852 8.420 8.585 4.651 4.619 9.027 9.195 9.377 9.047 4.518 4.782 4.793 4.724 4.891 4.884 7.060 6.696 4.584 4.753 4.939 4.956 9.931 10.057 4.961 4.662 5.287 VIC 560 nm 8.078 7.945 8.133 8.109 7.938 8.148 10.199 10.388 7.858 7.770 9.633 9.787 9.488 9.136 7.611 8.003 8.081 7.958 8.307 8.276 9.467 9.282 7.744 8.034 8.351 8.373 9.252 9.343 8.518 7.994 8.470 samples 1: 1304-776710 2: 1304-776710 3: 1330-641222 4: 1330-641222 5: 1431-673876 6: 1431-673876 7: 1468-919815 8: 1468-919815 9: 1481-343610 10: 1481-343610 11: 1525-402499 12: 1525-402499 13: 1536-037263 14: 1536-037263 15: 1544-581968 16: 1544-581968 17: 1656-254784 18: 1656-254784 19: 1658-020564 20: 1658-020564 21: 1671-950406 22: 1671-950406 23: 1703-964800 24: 1703-964800 25: 1704-902480 26: 1704-902480 27: PC 28: PC 29: NG 30: NG 31: RS

Multiplex Analysis
Different dyes for each target (Example: FAM, TET, VIC and JOE)
Real-time detection of four different retroviral 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 nucleotide sequences and four molecular beacons, each specific for one of the four amplicons and labelled with a differently coloured fluorophore. HIV-1: Fluorescein HIV-2: Tetrachlorofluorescein HTLV-1: Tetramethylrhodamine HTLV-II: Rhodamine Vet JA et al. PNAS (1999)

Summary
- Real Time PCR is Quantitative and

Qualitative Technique ( has wide range of applications) -Real Time PCR is Highly specific and sensitive rather than basic PCR -Low possibility of contamination (close tube system) - Less time consuming and less effort (amplification and detection at the same Time). - Expensive and need well trained people.

Thank you