Principle of Real-Time qPCR AND Applications Han-Oh Park, Ph.D. President & CEO BIONEER CORPORATION.

Principle of Real-Time qPCR AND Applications Han-Oh Park, Ph.D. President & CEO BIONEER CORPORATION

- - Principle of Real-Time Principle of Real-Time qPCR AND Applications qPCR AND Applications Exicycler Exicycler Understanding Real Time PCR Data

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Principle of Real-Time qPCR

Principle of PCR Target DNA Basics of PCR Heating 95 Cooling 55 Polymerase Primer Extension 72 Cycling 1 Cycle

Principle of PCR Cockerill FR III. Arch Pathol Lab Med. 2003;127:1112

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

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

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

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

Nigel Walker, NIEHS Principle of Real-Time PCR

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

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

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

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

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

Applications of Real-Time PCR

Real-Time PCR Applications Clinical Diagnostics - Bacterial/ Viral pathogen detection - Absolute pathogen quantification Drug therapy efficacy / drug monitoring Differential gene expression RNAi - siRNA validation SNP Genotyping Others - Food pathogen testing - Forensic studies

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

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.

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

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

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

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)

Experiment Design (Real-Time PCR /Relative quantification) Cell line A mRNA Cell line A + Drug mRNA Actin Target Relative Quantification (Real-Time PCR)

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

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

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

Allelic Discrimination Assays (Single Nucleotide Polymorphisms) G G C C T T A A SNP Genotyping (Real-Time PCR)

A A A T T T G G G C CC

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

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)

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

Special Features of Exicycler TM

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

1)Superior optic module -Light pipe technology (patented) 2)Light source -Short Arc Lamp -Life: 3,000 hrs -High Power, high quality, low maintenance 3)Excitation filter -Six position (blank 1, filter 5) -Wavelength range: 490-640nm -5 multiplexing 4) Emission filter -Six position (blank 1, filter 5) -Wavelength range: 520-670 nm -5 multiplexing 5)Powerful 2D CCD detector -High resolution and great sensitivity -Resolution : 325K pixel 6)Thermal gradient Characteristics of Exicycler TM

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.

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

Optic module of Exicycler TM Light pipe Camera lens Lamp

Comparison of Optics ExicyclerOther Company More homogeneous light intensities among wells => Reliable results without using reference dye

Comparison of Optics Exicycler Other Company Without reference dyeWith reference dye Without reference dye

Diversity of fluorescence dyes Excitation/Emission wavelength PositionExcitationEmissionDye 1490520Fluorescein, FAM, SYBR Green 2520550JOE, TET 3550580TAMRA, CY3 4580610Texas Red, ROX, RED610 5640670CY5, RED 670

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

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

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

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

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)

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

Experimental Results in Exicycler TM SYBR Green I data Without reference dye

Experimental Results in Exicycler TM TaqMan data (FAM) Without reference dye

Understanding Real Time PCR Data

Refreshing Logarithms and Exponentials 1 2 4 816 3264 t = 0123456

Refreshing Logarithms and Exponentials

f(x) = b x f(x) = log b x

Refreshing Logarithms and Exponentials For f(x)=bx a a > 1 a = 1 a < 1a < 0 a > 1

log-log plot Semi-log plot Regular plot

PCR reagent is the limiting factor! Copies of DNA=2 N

PCR Phases Log [DNA] Cycle # Exponential Plateau Linear Ethidium-Geldetection

Variable Linear Phase

Plateau Effect

SERIES OF 10-FOLD DILUTIONS

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

SERIES OF 10-FOLD DILUTIONS

Anatomy of an Amplification Plot 010203040 cycle number RnRn CTCT Threshold R n Sample No Template Control (NTC) Baseline

Basic Knowledge on Statistics 79 Normal distribution is the mean is the standard deviation Variance is

The standardized value of x is defined as It is also called a z-score.

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:

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.

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%

Confidence Interval So, we need to count the number of standard deviations from the mean But we dont know .

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).

t-statistic t = Sample mean Population mean s is the sample standard deviation

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).

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.

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.

Baseline Set Too Low

Baseline Set Too High

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

Log phaseLevel off/ plateau 32 16 8 4 2 DNA copy number (log) PCR cycle (Ct)

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.

Types of Quantification End-point quantification SNP typing Pathogen detection Absolute quantification Relative quantification Melting Curve (for Cyber Green I)

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

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 ) =

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) =

Comparative C T ( C T ) Method with Efficiency Correction

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

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

SNP Typing Using TaqMan One reporter for each allele k-means clustering Using Cyber Green I Primers with different lengths Melting curve examination

Thank you !

Principle of Real-Time qPCR AND Applications Han-Oh Park, Ph.D. President & CEO BIONEER CORPORATION.

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