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Slide 1
Principle of Real-Time qPCR AND Applications Han-Oh Park, Ph.D.
President & CEO BIONEER CORPORATION
Slide 2
- - Principle of Real-Time Principle of Real-Time qPCR AND
Applications qPCR AND Applications Exicycler Exicycler
Understanding Real Time PCR Data
Principle of PCR Target DNA Basics of PCR Heating 95 Cooling 55
Polymerase Primer Extension 72 Cycling 1 Cycle
Slide 11
Principle of PCR Cockerill FR III. Arch Pathol Lab Med.
2003;127:1112
Slide 12
Basics of PCR 1 Cycle 2 Cycle 3 Cycle N Cycle ? Ideal graph
Real graph Disadvantages of PCR End-Point PCR analysis is not
quantitative
Slide 13
Disadvantages of 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
Slide 14
Real-Time PCR Real-time PCR monitors the fluorescence emitted
during the reaction as an indicator of amplicon production at each
PCR cycle (in real time) as opposed to the endpoint detection
Slide 15
Fast, Accurate and Quantitative Results 2. Optical Components
3. Thermal Cycler 1. Fluorescence Dyes Key components of Real-Time
qPCR Principle of Real-Time PCR Real- Time qPCR
Slide 16
Nigel Walker, NIEHS Principle of Real-Time PCR
Slide 17
Real-Time PCRPCR SensitivityHighLow SpecificityHigh -use
specific probes Low -only size discrimination Quantitative results
Yes -Specific fluorescence No -EtBr staining Detection method
Probe-specific Fluorescence Agarose gel Electrophoresis Detection
rangeWide rangeShort range (
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 the reporter during extension. 3)The TaqMan assay accumulates
a fluorescence signal. 2) Annealing 1) Denaturation 3) Extension Q
F FQ Taq F Q F Q TaqMan probe TaqMan Probe
Slide 26
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
Slide 27
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, the
structure unfolds, separating the quencher from the dye and
allowing fluorescence. 2) Annealing 1) Denaturation 3) Extension Q
F Q Taq F Q F Molecular Beacon Molecular Beacon Probe
Slide 28
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
Slide 29
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 FF
Taq F F FF Energy transfer Hybridization Probe (FRET) FRET
Probe
Real-Time PCR Applications Structural Assay Uses DNA, typically
genomic extractions Single Nucleotide Polymorphism Functional Assay
Uses RNA extractions Uses reverse transcriptase to generate cDNA
templates Differential expression Diagnostics involving gene
expression RNA interference Clinical Diagnostic Assay Uses DNA or
RNA extracted from patients samples Viral/bacterial pathogens
Slide 33
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.
Slide 34
Detection of specific genes in pathogens (virus, bacteria,
fungi, etc) isolated from patients samples Quantification: copy
numbers of infected pathogens Therapeutic Drug Monitoring/Screening
Varicella-Zoaster Virus Real-Time PCR 10 6 copies Absolute
Quantification Pathogen Detection/Clinical Diagnostics
Slide 35
The Ct value correlates strongly with the starting copy number.
It is linear with the log of starting DNA concentration. Ct value
Absolute Quantification Sample 1 Ct:14 Conc: 2,500copy Threshold
Absolute Quantification
Slide 36
Compares transcriptional levels of genes between control and
experimental samples - Tissue distribution of target gene - Drug
screening/Drug efficacy - Gene expression profiling after drug
treatment Endogenous controls are used to normalize the data
Endogenous controls are genes common to both control and
experimental samples that do not change their expression levels
under the experimental conditions - GAPDH - -Actin - 18s ribosomal
subunit Differential Gene Expression Relative Quantification
Slide 37
Experiment Design (Traditional / Relative quantification) PCR
using same amount of mRNA after spectrophotometer quantification
ERROR Cell line A mRNA Cell line A + Drug mRNA Relative
Quantification (PCR)
Slide 38
Experiment Design (Real-Time PCR /Relative quantification) Cell
line A mRNA Cell line A + Drug mRNA Actin Target Relative
Quantification (Real-Time PCR)
Slide 39
ActinTarget Actin Target A > B (2 1 : 2 times much) Sample A
: 16 Ct Sample B : 17 Ct => Delta Ct = 1 Ct A > B (2 4 : 16
times much) Sample A : 17 Ct Sample B : 21 Ct => Delta Ct = 4 Ct
Relative Quantification (Drug-induced gene expression) Control cell
lines + Drug A Drug A treatment induced decreased target X gene
expression Relative Quantification
Slide 40
Relative Quantification (Tissue distribution / Delta Ct
Methods) Brain tissue Liver tissue Actin Target X Actin : 17 Ct
Target : 21 Ct => Delta Ct = 4 Ct Actin : 22 Ct Target : 26 Ct
=> Delta Ct = 4 Ct Target gene expression level between brain
and liver tissue is same Relative Quantification
Slide 41
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 10 8 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 SNP Genotyping
Slide 42
Allelic Discrimination Assays (Single Nucleotide Polymorphisms)
G G C C T T A A SNP Genotyping (Real-Time PCR)
Slide 43
A A A T T T G G G C CC
Slide 44
RNA interference: double stranded RNA (siRNA) forms a complex
(RISC) that binds to target mRNA and leads to its degradation
Target gene expression can be quantified by Real-Time PCR RNAi:
siRNA validation
Slide 45
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)
Slide 46
Improving Reproducibility Use clean bench (hood) Use aerosol
resistant tips Use calibrated micropipettors Use large volumes (5L
and up) Pipette into each reaction vessel once Cycle Good
TechniquePoor Technique Same Reagents, Different Results
Slide 47
Special Features of Exicycler TM
Slide 48
The Exicycler TM is a real time PCR system developed by
Bioneer. The Exicycler TM is equipped with an optical system that
fits above the thermal cycler. It readily utilizes most fluorescent
dyes, providing the widest choice of excitation / emission
wavelengths. It can also be used as a standard thermal cycler for
general PCR reactions. Real Time Quantitative Thermal Block
Exicycler
Superiors of Exicycler TM Analysis can be viewed in real time.
Melting curve can also be viewed in real time. Thermal gradient is
compatible. Range of fluorophore excitation and emission is 490~670
nm. It can be used for multiplexing with 5 different fluorophores.
Reliable results without using reference dye. Not necessary to
decontaminate the sample block. 96 samples can be tracked
simultaneously. All data is accessible as Excel file and jpg image.
Result reports, everyone can open on internet explorer. Software is
friendly to beginner at qPCR. USB communication interface. Auto
loading.
Slide 51
Patents of Exicycler TM A company BIONEER Intensity Profile at
Sample Reactor(Patents) 1) Real time monitoring apparatus for
amplified nucleic acid product -Appl. Con. : ROK -Appl. Date: Jun.
18, 2002 -Appl. No. : 10-2002-0033965 2) Real time monitoring
apparatus -Appl. Con. : ROK -Appl. Date : Apr. 3, 2003 -Appl. No. :
10-2003-0021145
Slide 52
Optic module of Exicycler TM Light pipe Camera lens Lamp
Slide 53
Comparison of Optics ExicyclerOther Company More homogeneous
light intensities among wells => Reliable results without using
reference dye
Slide 54
Comparison of Optics Exicycler Other Company Without reference
dyeWith reference dye Without reference dye
Slide 55
Diversity of fluorescence dyes Excitation/Emission wavelength
PositionExcitationEmissionDye 1490520Fluorescein, FAM, SYBR Green
2520550JOE, TET 3550580TAMRA, CY3 4580610Texas Red, ROX, RED610
5640670CY5, RED 670
Slide 56
Classified by light sources Laser High cost and high power
Small interference High maintenance cost Limited number of colors
(Laser based excitation limits fluorophore range) Can only be used
for well scan type (ABI 7900) LED Low cost and low power More
number of colors Inadequate for high throughput system Normally
used for well scan type (sequential data acquisition) Chromo4,
LightCycler, Opticon series, Rotor-Gene
Slide 57
Lamp Suitable for multi-color Unique solution for 2 dimension
sensor (Simultaneous data acquisition) High cost optic component
ABI 7500, 7300, iCycler series, LC480, Exicycler Classified by
light source Suitable for high throughput system
Slide 58
Classified by sensor and thermal cycling Point sensor (PMT,
photodiode) and rotating mechanism Low price and high temperature
uniformity Difficult to handle, cant support storage function No
system modularity Air heating not as accurate as Peltier
LightCycler (discontinued), Roter-Gene No gradient function to aid
in optimization Special consumables and loading block are
required
Slide 59
Classified by sensor and thermal cycling 2D sensor (CCD)
Simultaneous detection (suitable for high throughput) Increased
material costs, hard to calibrate ABI 7500, ABI 7300, iCycler,
LC480, Exicycler Point sensor (PMT, photodiode) and scanning
mechanism Cheap, simple, easy to calibrate Time lag due to scanning
Chromo4, MX3005P High maintenance cost Longer running time
Slide 60
Market trend 2D CCD is the current main stream for sensor ABI
7500, ABI 7300, iCycler, Roche LC480 High throughput Must support
plate format Bioneer is developing 1536 well format real time PCR
currently Powerful and various analysis software SNP typing,
Absolute & Relative Quantification, Infection Assay (using
internal control)
Slide 61
Comparison NameiCyclerSmartCyclerRotorGene Lightcycler 2
ABI7500Opticon 2Exicycler Company Bio-radCepheidCorbettRoche
Applied biosystem MJ researchBioneer Detection Simultaneous
Sequential SimultaneousSequentialsimultaneous Type 96 well16
tube32, 72 tube32 capillary96 well Gradient OxOO Light source
Tungsten Halogen Lamp LED Blue LED Tungsten Halogen Lamp LED Short
Arc lamp Detector 10 bit CCD Silicon Photodetector PMT Photo-
hybrid 10 bit CCDPMT 16 bit CCD Ex /Em range 400~700 450~650
470~660 470~710 488~650 470~700 490~670 Multiplex 4 color 6 color5
color2 color5 color
Slide 62
Experimental Results in Exicycler TM SYBR Green I data Without
reference dye
Slide 63
Experimental Results in Exicycler TM TaqMan data (FAM) Without
reference dye
Slide 64
Understanding Real Time PCR Data
Slide 65
Refreshing Logarithms and Exponentials 1 2 4 816 3264 t =
0123456
Slide 66
Refreshing Logarithms and Exponentials
Slide 67
f(x) = b x f(x) = log b x
Slide 68
Refreshing Logarithms and Exponentials For f(x)=bx a a > 1 a
= 1 a < 1a < 0 a > 1
Slide 69
log-log plot Semi-log plot Regular plot
Slide 70
PCR reagent is the limiting factor! Copies of DNA=2 N
Slide 71
PCR Phases Log [DNA] Cycle # Exponential Plateau Linear
Ethidium-Geldetection
Slide 72
Variable Linear Phase
Slide 73
Plateau Effect
Slide 74
SERIES OF 10-FOLD DILUTIONS
Slide 75
Effects of Efficienc y AFTER 1 CYCLE: 100% => 2.00x 90%
=> 1.90x 80% => 1.80x 70% => 1.70x AFTER N CYCLES: fold
increase = (efficiency) n
Slide 76
Slide 77
SERIES OF 10-FOLD DILUTIONS
Slide 78
Anatomy of an Amplification Plot 010203040 cycle number RnRn
CTCT Threshold R n Sample No Template Control (NTC) Baseline
Slide 79
Basic Knowledge on Statistics 79 Normal distribution is the
mean is the standard deviation Variance is
Slide 80
Slide 81
Slide 82
The standardized value of x is defined as It is also called a
z-score.
Slide 83
Central Limit Theorem A very important result in statistics
that permits use of the normal distribution for making inferences
(hypothesis testing and estimation) concerning the population mean.
If a variable x (with any distribution) has a population mean and
standard deviation , then: the distribution of sample means (from
samples of size n taken from the population), has the following
distribution as n tends to infinity:
Slide 84
Confidence Interval for the Mean A way of expressing the
uncertainty in as an estimate of . 95% confidence interval says
that on average 95 % of the time, if you estimate an interval for
this way, the true value of will be inside the interval.
Slide 85
Common Z levels of confidence Commonly used confidence levels
are 90%, 95%, and 99% Confidence Level Z-value (Z-score, Critical
value) 1.28 1.645 1.96 2.33 2.58 3.08 3.27 80% 90% 95% 98% 99%
99.8% 99.9%
Slide 86
Confidence Interval So, we need to count the number of standard
deviations from the mean But we dont know .
Slide 87
When is unknown In most cases knowledge of the true variability
of the measurement will not be available. In these cases, we
proceed by substituting with the sample standard deviation s: And
basing our inference on the t distribution with n-1 degrees of
freedom (where n is the size of the sample).
Slide 88
t-statistic t = Sample mean Population mean s is the sample
standard deviation
Slide 89
The t distribution The t distribution is symmetric, and
centered around zero. It has fatter tails compared to the standard
normal distribution. The t distribution is defined by n-1 degrees
of freedom (n is the sample size).
Slide 90
Degrees of Freedom Essentially the number of independent pieces
of information provided by the sample. Initially, every sample has
n independent pieces of information (as many as the number of
observations). If we know the first n-1 observations, we can
compute the n th one, and thus there are n-1 independent pieces of
information.
Slide 91
Baseline Background or noise signal is often normally
distributed. Signal is meaningful only if it is higher enough than
the background signal. Baseline value is a single value or a
function to represent the background signal. Baseline is the cycles
used to calculate the baseline value.
Slide 92
Baseline Set Too Low
Slide 93
Baseline Set Too High
Slide 94
Threshold At least 5 to 10 z-value higher than the mean of the
distribution of background signal. Many competing factors:
background noise, stabilized regions in exponential phase for all
replicates, maximizing sensitivity
Slide 95
Log phaseLevel off/ plateau 32 16 8 4 2 DNA copy number (log)
PCR cycle (Ct)
Slide 96
Rn Rn (normalized reporter) is the fluorescence emission
intensity of the reporter dye multiplied by the calibration factor.
Rn + is the Rn value of a reaction containing all components (the
sample of interest); Rn - is the Rn value detected in NTC. Rn is
the difference between Rn + and Rn -. It is an indicator of the
magnitude of the signal generated by the PCR. Rn is plotted against
cycle numbers to produce the amplification curves and gives the C T
value.
Slide 97
Types of Quantification End-point quantification SNP typing
Pathogen detection Absolute quantification Relative quantification
Melting Curve (for Cyber Green I)
Slide 98
Absolute Quantification Absolute/Relative quantification Used
serial diluted standards of known concentration to generate a
standard curve. Standard curve is a linear relationship between the
c t and the initial amount amounts of RNA or cDNA. This allows the
determination of the concentration of unknowns based on their c t
values Assumes that all standards and samples have equal
amplification efficiencies. The concentrations of serial dilutions
should encompass all samples and stay within the range that can be
quantified Rep 1 0 hrs 12 hrs pi 24 hrs pi Rep 2 0 hrs 12 hrs pi 24
hrs pi Rep 3 0 hrs 12 hrs pi 24 hrs pi Pooled sample Dilution 1 (10
-1 ) Dilution 2 (10 -2 ) Dilution 3 (10 -3 ) Generating a standard
curve Carry out runs in triplicates
Slide 99
Comparative C T ( C T ) Method C T (target gene, control) C T
(endog. refer. gene, control) = C T,cont (control tissue) C T
(target gene, exp.) C T (endog. refer. gene, exp.) = C T,exp
(experimental tissue) C T, exp - C T, cont = C T Target gene exp
Target gene cont 2 (average C T ) =
Slide 100
Comparative C T ( C T ) Method with Efficiency Correction C T
assumes similar efficiencies Target gene exp Target gene cont (E
target ) C T target (cont - exp) (E cont ) C T ref (cont - exp)
=
Slide 101
Comparative C T ( C T ) Method with Efficiency Correction
Slide 102
Exist/Non-Exist Assay Internal Positive Control (IPC) Four
types of reactions 1. No Amplification Control (NAC) blocked IPC,
to calculate IPC threshold 2. No Template Control (NTC) to
calculate target threshold 3. Target 4. Unknown sample
Slide 103
Exist/Non-Exist Assay Statistical methods for choosing
thresholds Three possible outcomes 1. Target below threshold, IPC
above threshold Minus 2. Target above threshold, IPC above
threshold Plus 3. Target below threshold, IPC below threshold
Undetermined
Slide 104
SNP Typing Using TaqMan One reporter for each allele k-means
clustering Using Cyber Green I Primers with different lengths
Melting curve examination