PCR - BTBS
Contents
1. T1 T2 T3 T4 Gene expression changes during differentiation Differentiation protocol Collect Total RNA at different time points Measure 1 HKG and 1 GOI TNFa Repeat experiment 3x biological replicates Sample to Insight 29899 qPCR for gene expression 00000 QIAGEN Work Flow Gene expression profiling Total RNA Y Y Y Assay Assa y Thermocycling y Data Analysis Sample to Insight 7 asses Factors Critical For A Successful qPCR Assay DNAor RNA sample preparation Template quality Appropriate sample prep kits reagents Inhibitors can compromise RT or PCR Reverse transcription to convert RNA to cDNA Choose RT kits type of RT which type of primers controls Assay design chemistry specificity PCR efficiency amp throughput amp cost Choose validated assay or need to validate our own Running PCR Commercial mastermix or make own primer probe master mix Data analysis tool User friendly Streamlined data analysis module Sample to Insight 7 asses RNA purity and integrity RNA Isolation Qiazol Column based method RNeasy Both Efficient lysis and inhibition of RNases molecular grade RNA MIRNA Use a kit specific for miRNA and mRNA miRNeasy mini Kit Qiazol phenol guanidine based lysis Instant inactivation of RNases Instant end of biological activities Column cleanup Molecular biology grade2. Polymerase MH dNTPs ara Primers 2 qPCR in Action Polymerase N k A AR Polymerase art iets AO DNA Template ss or ds Polymerase MH dNTPs ya Primers 2 3 Extension 60C Sample to Insight qPCR in Action Polymerase EA ECOS ek se DNA Template ISS Or AS Polymerase Polymerase aan dNTPs Primers 2 3 Extension 60C Pee Sample to Insight 99999 qPCR in Action QIAGEN gt 41 j O Ee ee eee o A bi O A A Ak DNA Template d ss or ds Ames Polymerase E dNTPs yn Primers 2 2 a 4 Repeat 95C prum cano Sample to Insight 99999 qPCR in Action IAGEN gt 1 a a E e E EE E snap OOD e DNA Template O How do you make this a quantitative PCR Measure DNA amount at end of each cycle to get ratio of DNA or absolute amount if using a Bolmerace standard atap dNTPs o ara Primers 2 4 Measure amount of PCR Product E Sample to Insight 20060 Reporter chemistries QIAGEN Real Time qPCR Fluorescence Chemistry DNA binding agents o SYBR Dye Hydrolysis Probes n Dual labeled Hydrolysis Tagman probe Others such as hybridization probes o Molecular beacon and scorpion probes Sample to Insight 29089 Reporter chemistries SYBRO Green Assay 060009 QIAGEN Non fluorescent SYBR Ga a o o o SYBR I binds
3. 2 44 Sample to Insight _ 3883s Data amp analysis Delta Delta Ct Method Amplification Plots SS GAPDH control R GAPDH treatment mea TNFa control INFo treatment GOI analysis line 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle number AACt ACt TNFa ea GAPDH reat Act TNFO ontro GAPDH The fold change 24400 Sample to Insight al Data amp analysis 17 1 17 2 17 2 aPCR replicates 1 Average Ct values for all gene replicates 2 Calculate Delta Ct value GOI HKG 13 8 14 3 14 4 3 Average Delta Ct values between experiments replicates Average 14 17 4 Calculate Delta Delta Ct values Delta Ct experiment Delta Ct control DD Ct 5 00 5 Calculate Fold Change 2 Delta Delta Ct TNFa is up regulated 32 fold in the treated cells versus the control Sample to Insight Resources Support About QIAGEN Careers Ses a Hello Christine Davis Y Order TA Wishlist Cd Contact us Products Genes amp Pathways Research Portais Technology Portais Data Analysis Center Easily interpret results The GeneGlobe Dats Analysis Center is a web resource for scientists analyzing their real time PCR or NGS dats The real time PCR modules transform threshold cycle Cr values to calculated results for gene and miRNA expression somatic mutation detection and copy number measurements The NGS module supports the analysis of QIAGEN s GeneRea
4. 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 CYCLE NUMBER Figure 4 RT Profiler PCR Arrays and RT qPCR Assays Have Sufficiently Wide Dynamic Ranges Sample to Insight asses Characteristics of a good qPCR Assay Sensitivity Sensitivity How many copies can my assay detect Important for low expressed genes or where there is limited sample Two Methods Method 1 Use primers to make PCR product T A clone grow up isolate quantitate and use for qPCR reactions Method 2 Use gDNA as template and use mass of gDNA to calculate copy number and assume 1 target per genome or actually calculate targets using bioinformatics 22982 Characteristics of a good gPCR Assay Specificity QIAGEN Specificity 1 target amplified Two Methods SINGLE DISSOCIATION CURVES Melt Curve analysis 1 peak 1 product Agarose gel Band at correct size rn Re Ko N u a w Y tj z um BMP4 BMPS AGAROSE GEL Sample to Insight 7 22352 Characteristics of a good gPCR Assay Specificity Melting Curve Analysis Plot Normalized Reporter The General Program Steps FE Pe Haa Heat to 94 to denature DNA LASG e PPK Cooling to 60 to let DNA bo Sart n E SNS double strands anneal 3 SORA SON Slowly heat increase temp to Q Te e i 2 E AH 0 2C sec while plotting the O a PRH fluorescent signal vs E EA 50 fluorescence temperature q
5. pe gt lt drop AN As the temp increases DNA 2 ny E na melts fluorescent signal should O 1 ON 2 AA decrease L ERCE o j aW 85 0 Significant drop in signal when Gene A Tm 77 36 Gene B Tm 78 94 See aN incite Temperature Sample to Insight 7 29352 Characteristics of a good gPCR Assay Specificity Melting Curve Analysis Plot 15 negative Derivative Reporter Single melt curve of each amplicon is required for specificity validation Derivative Reporter Rn delta F delta T the change rate Fi 65 0 70 0 75 0 80 0 85 0 90 0 25 0 Tm 78 74 Temperature C Temperature Sample to Insight 29300 Biological replicates are better than technical replicates 00000 QIAGEN Biological Replicates 3 e different experiments a Shows variability due to experiment is Technical replicates 3 different measurements for same step y Shows variability due to pipetting machine enzymes etc y 10075x 0 2891 R 0 989 Sample to Insight 99999 Analyzing qPCR curves How to Define Baseline QIAGEN Linear Amplification Plot og Automated Baseline Option m A if an instrument has a adaptive baseline ng function 05 3 Manual Baseline Option a 1 Use linear view of the plot 03 2 Set up the baseline reading from 02 A cycle 2 to the cycle that 2 cycles before 01 il Ct the earli
6. to double strand DNA but not s a denaturation single strand DNA Little fluorescence emitted CN O E a from SYBR I in solution o img ae SYBR upon binding to double strand DNA c o a Y gt primer annealing Ee Se emits fluorescence very brightly o a Fluorescent SYBR The SYBR signal intensities correlate with DNA amplified amplicon amount thus the initial sample input amounts NA EME NAH RE H extension BEE HERE HE ES RARE e Simple amp cost saving High Specificity Is Required when using SYBR Green since SYBR binds all double strand DNA non specific or primer dimer Sample to Insight Fluorescence Signal r Fa EM Sample to Insight Reporter Chemistries Understanding Kinetics in PCR Amplification Plot Linear scale EE AA A US 2404004001024 LAS TA Cycle End point PCR data collection at plateau gel analysis Reactions start varying due to reagent depletion amp decreased PCR efficiencies enzyme activity more product competing for primer annealing Real time PCR does early phase detection at the exponential state Precisely proportional to input amounts 22888 Reporter Chemistries Hydrolysis Based Probe Tagman Probe Assay Fluorophore Quencher Forward PCR primer Nee The fluorescence of the reporter dye is suppressed by the quencher Reverse PCR primer Amplification Assay Polymerization a
7. 298909 Introduction To Real Time Quantitative PCR qPCR 00000 QIAGEN Webinar related questions Technical Support QlAwebinars QIAGEN com BRCsupport giagen com Sample to Insight 29080 Welcome to our 3 part webinar series on gPCR 00009 QIAGEN D qPCR technology overview applications data a analysis and interpretation Q Part 1 Introduction to Real Time PCR Q PCR qPCR art PCR U Part 2 Pathway focused Gene Expression Analysis Advanced Real Time PCR Array Technology U Part 3 PCR Array Data Analysis Tutorial Sample to Insight 806888 29389 Legal Disclaimer QIAGEN an AT A a ea ie aa Ap ma ip E j i QIAGEN products shown here are intended for molecular jasa applications These products are not intended for the diagnosis prevention or treatment of a disease For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www QIAGEN com or can be requested from QIAGEN Technical Services or your local distributor Sample to Insight 29889 How does qPCR work 009009 QIAGEN Question How far apart are the 2 cars Cars race at same speed to finish line As car 1 crosses finish line calculate time for car 2 to finish Calculate difference in starting position mathematically d rate x time Sample to Insight How does
8. HAVHOHOS L Q GQHHOOHHAGQHHHOHS aDOVOVODARARDADO EII IO 7 O QQOGHOHGHHHHWHHHHH 2OQH DQHHOHOHD HHHHS OGCOCOOODOOCODOOOOQG OPOOOGOOOOOOOOOOO OOONOOOOOCOOOOOO G QHQGQHOOOHHHDDOHO 9 HQHGHHHOHHHDWBOQOHO D G9 QQSGHVDHHHDDOHOHH B QHG HHHADWDHOOOS qavawwOre nanwHEZ00a Positive PCR controls Reverse transcription controls Genomic DNA control Housekeeping genes GDC RTC PPC HKG Pre validated qPCR assays with controls Sample to Insight 299909 Thank You for Attending 00000 QIAGEN Questions Ask now or contact Technical Support e Call 1 800 426 8157 e Email BRCsupport QIAGEN com Sample to Insight
9. NA 18 Biomarker discovery in biofluids Maating the challanges of biomarker research Research solutions for liquid biopsies 12 Genomic biomarker discovery 19 Circulating biomarkers New solutions for DNA and RNA 26 Critical factors for successful RT PCR IncRNAs in cancer amp miRNA regulation 13 New advances in IncRNA research qPCR introduction 20 PCR arrays for pathway analysis 27 Optimize NGS conditions based on your DNA sample PCR array dota analysis tutorial 9 30 am EST 2 30pm GMT 1 00 pm EST 6 00pm GMT 11 00 am CST Beijing Time Register http www giagen com Knowle dge and Support Webinars 384 Well Upcoming Webinars Still searching gene by gene Learn about RT Profiler PCR Arrays Catalogued RT Profiler by pathway amp disease L OHDOHDOOHDAD DOHOOS RH BOQOHDDD DODOH HHIDOS L QQ DQHEODO OHOHADDO DGOS 1 QOQGQH OHOHAADOOSS 100008026060606068 0990850 0O10F0A00 G HD QHAQOHOHHDOHHDSOS DODVOPHPPANHHODO DOOM DADAHOO DOVOBOCODARAHHO 9 OOQHAD GOOHDADDOAGQS OVO 508 qavawwore sa sFEZO0a Asas Cr s 8 m S28 338282288 oF FS PR SSE os FS 888 E nossas mr E 082393328 ms PRs 2 KD S 2a es 328 os FR Ss 3R ns sa 8 2 2 s2a25 83 2 qaavuaweort Positive controls Reverse transcription PCR controls Genomic DNA control Housekeeping genes J B QHDDODOHOVDDHOAVHOS RGQOHGQHHHOQOHH
10. RNA Archive miRNA for next project Sample to Insight 20060 0009 QIAGEN RNA purity and integrity Purity Quantity Spectroscopic measure 260 280 and 260 230 eOD260 is used to calculate amount of nucleic acid 260 280 ratio typical minimum value 1 8 2 0 260 230 ratio typical minimum value 1 7 Low ratio may indicate a contaminant protein QIAzol Carbohydrates Glycogen Absorbance measurements do not show integrity of RNA Integrity Denaturing RNA Agarose Gel QlAxcel system Agilent 2100 Bioanalyzer Usually through ribosomal bands o m a QlAxcel Bioanalyzer Capillary electrophoresis Automate RNA integrity analysis RNA integrity analysis number Sample to Insight Factors Critical For A Successful qPCR Assay qPCR Components A Templates B Primers Probes RNA Starting amount 10 1000 copies C Master Mix of NA per qPCR assay DNA polymerase For a low expressed gene need Mg 10ng equivalent of RNA per dNTPs reaction Buffer Want to start with about 100pg to Passive reference dye 1ug RNA Reverse Transcription One Step or Two Step Reaction e 1 Tube Reaction 2 separate reactions Transfer cDNA alquem e RT Reaction y o TU eS e qPCR Reaction Sample to Insight 99999 Reverse Transcription QIAGEN Reverse Transcription Used to make cDNA copy of RNA Reagents Reverse transcriptase many different kinds dNTPs Buf
11. _i v rr oso Ia a 2 Primer binding followed by extension e o eym Y Probe displacement and cleavage Probe cleavage by Tag to free the reporter dye thus the fluorescence intensity correlates with the initial Result E sample input amounts Fluorescence P 3 a o Taq has 5 gt 3 exonuclease activity 4 r 4 PCR Products Cleavage Products Each amplicon needs a seguence specific probe cost amp time Sample to Insight Fluorescence Signal r Fa EM Sample to Insight Reporter Chemistries Understanding Kinetics in PCR Amplification Plot Linear scale EE AA A US 2404004001024 LAS TA Cycle End point PCR data collection at plateau gel analysis Reactions start varying due to reagent depletion amp decreased PCR efficiencies enzyme activity more product competing for primer annealing Real time PCR does early phase detection at the exponential state Precisely proportional to input amounts 7 evoco Characteristics of a good qPCR Assay What factors do you need to address to create a good PCR Assay Amplification efficiency 100 during exponential phase template product doubles with each cycle Sensitivity Able to detect down to reasonable quantities of template in 1 reaction 10 50 copies Specificity 1 assay 1 target no off target amplification or primer dimers e Melt curve analysis 1 peak 1 product e Agarose gel Dynamic Range A
12. bility to detect genes with varied expression levels another judge of sensitivity e 10 to 10 copies is ideal Reproducibility Confidence in your results enables profiling of multiple genes in the same sample All lab members get the same results Technical reproducibility ensures changes seen in results are due to the biology and not the technology itself or sample handling Sample to Insight coge Characteristics of a good qPCR Assay Amplification Efficiency 00000 QIAGEN aie Ca Amplification Efficiency reliable and accurate experiment Two Methods Standard curve X axis dilution 0 1 600 1 801 1 602 1 603 1 804 1 605 1 8206 1 807 1 808 1 609 1 610 a NUMBER OF GENE COPIES Y axis Ct value THRESHOLD CycLE C Amp efficiency 10 1 slope 4 100 RN VS CYCLE Single curve analysis TW PCR Miner Aa ta A E http miner ewindup info ver VA Gl Wl al wy son A f 1 AI di Sd DART www gene Tar i ie 18 20 22 24 26 28 50 32 38 36 38 M quantification de DART PC CYCLE NUMBER R version 1 0 xls R THRESHOLD CYCLE C Figure 4 RT Profiler PCR Arrays and RT qPCR Assays Have Sufficiently Wide Dynamic Ranges Sample to Insight Positive calls 4 100 Input RNA ng Human Inflammatory Cytokines amp Receptors RT Profiler PCR Array ig 5 TI y ia pi 8 18 49 19 208 2087 1 7 0 aot wo 1 A o amp y THRESHOLD CYCLE C a
13. d target enrichment panels Register on QIAGEN com to sccess this complimentary suite of tools to accelerate your data analysis and interpretation Learn more Demo gt Resources Choose format Experiment Performed Using Please choose Choose Array Choose Instrument Choose Data Analysis Type Specify CatNo Sample to Insight Investor Relations Press amp Media SiteMap Privacy Policy Trademarks amp Disclaimers Cnange Language Contact QIAGEN Contact Technical Service Contact Customer Care Sunshine Act More Contacte QIAGEN 2013 15 All rights reserved Sample to Insight cones Data Analysis Tools Resources Support About QIAGEN Careers rc Q Hello Christine Davis v Y Order Ly Wishlist CJ Contact us v Upload data gt Analysis setup Plots amp charts gt Export data gt What s next You Selected Instructions Technology RT Profiler PCR Array Catalog Number PAHS 021Z Plate Format 96 well Want to analyze data from a different product Return to GeneGlobe Data Analysis Center Fie No file selected File must be a MS Excel Sheet in XLS format not XLSX E Does your data look like this If not please download the appropriate Excel template to format your data correctly Excel Templates for Formatting your Experimental Data E B C D E E IResting6h Resting6h Resting6h 6 h Stimulation 6 h Stimulation Control Group Control Group Control Group Test Grou
14. est visible amplification Baseline Iei PE 3 Usually a baseline falls in 3 15 cycles 014000 ARIZA AAA Cycle Sample to Insight Analyzing gPCR curves How To Define Threshold Log View Amplification Plot maan Use log view of amplification plot Exponentia Threshold should be higher than remy baseline higher than the noise level 1 Threshold Threshold should at LOWER 1 3 or 1 2 Oh of the linear phase of amplification Linear phase exponential phase 0 001 A Different runs across samples for the a same experiments should have the iii same threshold for comparison a an a O IN gempa Ps GF ENNRUNHOKBARARANVQNAAA Cycle Sample to Insight E 33888 Data Analysis Housekeeping Reference Genes Any changes GOI A in control cells Ly GOI A in drug treated cells Reference gene Em Expression level remains consistent under experimental conditions different tissues E Aimed to normalize possible variations during E Sample prep amp handling e g use the same number of cells from a start E RNA isolation RNA quality and quantity E Reverse transcription efficiency across samples experiments E PCR reaction set up E PCR reaction amplification efficiencies Sample to Insight 22520 Data Analysis Commonly Used Housekeeping Genes QIAGEN Table 7 Housekeeping genes commonly used as endogenous references Gene symbol Relative expression level Gene Human Hu
15. fers for RT Primers Random pentamers or hexamers Oligo dT Both Control RNA to monitor reverse transcription kit Important Notes Ensure RT reaction is linear Do not try to reverse transcribe too much RNA Sensitivity of qPCR step is dependent on good RT reaction lt Sy q Monitor RT reaction to ensure equal RT efficiency across all samples ry d E Sample to Insight What is in a PCR Reaction PCR Polymerase Chain Reaction Exponential Amplification of DNA in single tube All reagents in excess non limiting Components Thermostable polymerase dNTPs Primers Template Sample to Insight qPCR in Action N T DNA Template ss or ds Polymerase ET dNTPs ara Primers 2 DNA Template ss or ds qPCR in Action A OOO Polymerase Le dNTPs ara Primers 2 age E 1 Heat denature template 95C 2 Annealing 60C 3 Extension 60C 4 Repeat 95C Sample to Insight qPCR in Action Heat denature DNA Template ss or ds Polymerase de dNTPs 1 Heat denature template 95C ara Primers 2 age E Sample to Insight eegee qPCR in Action QIAGE l DNA Template Polymerase RT dNTPs I AJ 2 Annealing 60C 5 Primers 2 age E Sample to Insight qPCR in Action ml Polymerase 2 Annealing 60C Sample to Insight DNA Template ss or ds
16. itial amount of target template Sample to Insight What is GPCR Applications and workflow Applications for qPCR ES Gene Expression Profiling Analysis cDNA analysis two step qRT PCR miRNA Expression Profiling Analysis TT RNA template cDNA template y v Transfer of cDNA amplifcation T qPCR products y ev y SNP Genotyping amp allelic discrimination gDNA analysis qPCR Somatic Mutation Analysis Copy Number Detection Variation Analysis Chromatin IP Quantification DNA DNA Methylation Detection re Pathogen Detection J N Amplification Viral Quantification Y y q QPCR products Sample to Insight 29929 What is GPCR Applications and workflow QIAGEN Work Flow A Brief Look Sample RNA total mRNA Sample QC Sample QC os rsa Uh Real Time PCR Set Up m Instrument Set up amp thermal cycling Data Output amp Analysis Sample to Insight _ 3883s What is qPCR Applications and workflow Applications for qPCR ES Gene Expression Profiling Analysis miRNA Expression Profiling Analysis SNP Genotyping amp allelic discrimination Somatic Mutation Analysis Copy Number Detection Variation Analysis Chromatin IP Quantification DNA Methylation Detection Pathogen Detection Viral Quantification Sample to Insight 20820 gPCR for gene expression Application example QIAGEN Osteogenesis Day 16 e hMSC T4
17. man Mouse 185 ribosomal RNA RRN18S Actin beta ACTB Glyceraldehyde 3 phosphate dehydrogenase GAPDH Phosphoglycerate kinase 1 PGKI Peptidylprolyl isomerase A PPIA Ribosomal protein L13a RPLI3A Ribosomal protein large PO RPLPO Acidic ribosomal phosphoprotein PO Beta 2 microglobulin B2M Tyrosine 3 monooxygenase tryptophan YWHAZ 5 monooxygenase activation protein zeta polypeptide Succinate dehydrogenase complex subunit A flavoprotein Fp Transferrin receptor Aminolevulinate delta synthase 1 Glucuronidase beta Hydroxymethylbilane synthase Hypoxanthine phosphoribosyltransferase 1 TATA box binding protein Tubulin beta Tubulin beta 4 Nya indicates relative abundance of the transcripts Sample to Insight Data amp analysis 1 Average Ct values for all gene replicates 2 Calculate Delta Ct value between GOI and HKG for each experiment 3 Average Delta Ct values between experiments replicates 4 Calculate Delta Delta Ct values Delta Ct experiment Delta Ct control 5 Calculate Fold Change 2 Delta Delta Ct Sample to Insight 29000 Data 4 analysis 00000 QIAGEN Normalized Gene Expression Level Any changes Target Gene A in control cells Target Gene A in drug treated cells ACt Ct Target A treated Ct Ref B treated ACt Ct Target A control Ct Ref B control gt AA Ct A Ct treated Ct control Normalized target gene expression level
18. p 1 Test Group 1 29 08 29 02 29 27 29 89 32 02 32 13 31 96 31 15 33 83 34 22 33 09 31 57 33 95 33 26 32 65 31 3 Undetermined Undetermined Undetermined Undetermined Undetermined 29 28 84 28 53 26 67 Undetermined 37 05 Undetermined 37 28 27 33 27 11 27 31 29 54 25 52 25 6 25 81 21 26 27 12 27 21 27 12 16 77 35 53 36 21 37 66 33 49 23 28 23 16 21 34 36 32 92 35 17 36 08 34 1 33 1 25 36 25 3 25 02 96 Well Cataloged PCR Array Sample to Insight Topics Covered Today 1 What is qPCR Applications and workflow 2 QPCR for gene expression What is the change in gene expression during differentiation Factors Critical For A Successful qPCR Assay RNA purity and integrity Reverse Transcription Reporter chemistries 3 4 5 6 qPCR in Action 8 Characteristics of a good qPCR assay 9 Analyzing qPCR curves 10 Data amp analysis Sample to Insight 2 Analysis somatic BRCA and BRCA2 varionts 9 Host pathogen interactions Microbiome from identification to characierization 23 Innate immune system 30 Toll like receptors in inflammation Sample to Insight Upcoming webinars Cancer stem cells NGS introduction to technology and applications 17 Addressing the challenges of NGS workflows Targeted NGS for cancer research 31 NGS data be for genetic profiling Exosomes in liquid biopsy 11 Maximize quantity and purity of exosomal R
19. qPCR work Question How far apart are the 2 cars Many cars how to differentiate cars of interest Sample to Insight 29889 How does qPCR work 009009 QIAGEN Question How far apart are the 2 cars Cars race at same speed to finish line As car 1 crosses finish line calculate time for car 2 to finish Calculate difference in starting position mathematically d rate x time Sample to Insight Seminar Topics 1 What is qPCR Applications and workflow 2 QPCR for gene expression What is the change in gene expression during differentiation Factors Critical For A Successful gPCR Assay RNA purity and integrity Reverse Transcription qPCR in Action Reporter chemistries Characteristics of a good qPCR assay e 2 NP DA SS Analyzing gPCR curves 10 Data amp analysis Sample to Insight What is GPCR Applications and workflow What does Real Time gPCR stand for Quantitative Polymerase Chain Reaction qPCR is a sensitive and reliable method for detection and quantification of nucleic acid DNA amp RNA levels it is based on detection and quantification of fluorescence emitted from a reporter molecule at real time This detection occurs during the accumulation of the PCR product with each cycle of amplification thus allows monitoring the PCR reaction during early amp exponential phase where the first significant increase in the amount of PCR product correlates to the in
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