2Real-Time PCR David A. Palmer, Ph.D. Technical Support, Bio-Rad LaboratoriesAdjunct Professor, Contra Costa College
3Objectives Today we’ll talk about Real-Time PCR: What is real-time PCR used for?How does real-time PCR work?What chemicals and instruments are used to detect DNA?What does real-time data look like?How can we demonstrate real-time PCR in the classroom?
4Part 1: What is Real-Time PCR and what is it used for?
5What is Real-Time PCR?PCR, or the Polymerase Chain Reaction, is a process for the amplification of specific fragments of DNA.Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses.As we will see, Real-Time PCR allows us to measure minute amounts of DNA sequences in a sample!
6Conventional PCR tells us “what”. Real-Time PCR tells us “how much”. What is Real-Time PCR?Conventional PCRtells us “what”.Real-Time PCRtells us “how much”.
7What is Real-Time PCR used for? Real-Time PCR has become a cornerstone of molecular biology. Just some of the uses include:Gene expression analysisCancer and Drug researchDisease diagnosis and managementViral quantificationFood testingPercent GMO foodAnimal and plant breedingGene copy numberForensicsSample identification and quantification
8Real-Time PCR in Gene Expression Analysis Example: BRCA1 Expression ProfilingBRCA1 is a gene involved in tumor suppression.BRCA1 controls the expression of other genes.In order to monitor level of expression of BRCA1, real-time PCR is used.Determine gene expression and publish scientific paper!Real-Time PCRDNABRCA1mRNAProtein
9Real-Time PCR in Disease Management Example: HIV TreatmentDrug treatment for HIV infection often depends on monitoring the “viral load”.Real-Time PCR allows for direct measurement of the amount of the virus RNA in the patient.Real-Time PCRViral RNAMeasure amount of virus, adjust prescriptions.
10Real-Time PCR in Food Testing Example: Determining percentage of GMO food contentDetermination of percent GMO food content important for import / export regulations.Labs use Real-Time PCR to measure amount of transgenic versus wild-type DNA.Real-Time PCRwt DNAGMO DNAInternational shipments depend on results!
11Real-Time PCR in Forensics Example: Real-Time PCR in Forensic Analysis!Stain Identification:New Real-Time methods can be directly used to identify the composition of unknown stains, with much better accuracy than traditional “color-change” tests.DNA Quantification:Since standard forensic STR Genotyping requires defined amounts of DNA, Real-Time PCR can be used to accurately quantify the amount of DNA in an unknown sample!What is it ??Enough DNA to ID ??
13How does real-time PCR work? To best understand what real-time PCR is, let’s review how regular PCR works...
14The Polymerase Chain Reaction How does PCR work?? 5’3’5’3’5’3’d.NTPs5’3’Primers5’3’Thermal StableDNA Polymerase5’3’5’3’5’3’5’3’Add to Reaction Tube5’3’5’3’5’3’Denaturation5’3’5’3’5’3’Annealing
15The Polymerase Chain Reaction How does PCR work?? 5’3’5’3’5’3’5’3’ExtensionTaq5’3’5’5’5’3’TaqExtension ContinuedTaq5’3’5’5’Taq3’Repeat
16The Polymerase Chain Reaction How does PCR work?? 5’3’Cycle 24 Copies5’3’Cycle 38 Copies
17How does Real-Time PCR work? …So that’s how traditional PCR is usually presented.In order to understand real-time PCR, let’s use a “thought experiment”, and save all of the calculations and formulas until later…
18Imagining Real-Time PCR To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…
19Imagining Real-Time PCR What’s in our tube, at cycle number 25?A soup of nucleotides, primers, template, amplicons (the amplified DNA product), enzyme, etc.1,000,000 copies of the amplicon right now.
20Imagining Real-Time PCR How did we get here? What was it like last cycle, 24?Almost exactly the same, except there were only 500,000 copies of the amplicon.And the cycle before that, 23?Almost the same, but only 250,000 copies of the amplicon.And what about cycle 22?Not a whole lot different. 125,000 copies of the amplicon.
21Imagining Real-Time PCR How did we get here? If we were to graph the amount of DNA in our tube, from the start until right now, at cycle 25, the graph would look like this:
22Imagining Real-Time PCR How did we get here? So, right now we’re at cycle 25 in a soup with 1,000,000 copies of the target.What’s it going to be like after the next cycle, in cycle 26?
23Imagining Real-Time PCR So where are we going? What’s it going to be like after the next cycle, in cycle 26?Probably there will be 2,000,000 amplicons.And cycle 27?Maybe 4,000,000 amplicons.
24Imagining Real-Time PCR So where are we going? What’s it going to be like after the next cycle, in cycle 26?Probably there will be 2,000,000 amplicons.And cycle 27?Maybe 4,000,000 amplicons.And at cycle 200?In theory, there would be 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons…Or 10^35 tons of DNA…To put this in perspective, that would be equivalent to the weight of ten billion planets the size of Earth!!!!
25Imagining Real-Time PCR So where are we going? A clump of DNA the size of ten billion planets won’t quite fit in our PCR tube anymore!!!Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out.So exponential growth does not go on forever!
26Imagining Real-Time PCR So where are we going? If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this:
27Imagining Real-Time PCR Measuring Quantities How can all this be used to measure DNA quantities??
28Imagining Real-Time PCR Measuring Quantities Let’s imagine that you start with four times as much DNA as I do.Picture our two tubes at cycle 25 and work backwards a few cycles.Cycle 25CycleMeYou251,000,0004,000,00024500,0002,000,00023250,000
29Imagining Real-Time PCR Measuring Quantities So, if YOU started with FOUR times as much DNA template as I did……Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would!Imagining Real-Time PCR Measuring Quantities
30Imagining Real-Time PCR Measuring Quantities What if YOU started with EIGHT times LESS DNA template than I did?Cycle 25CycleMeYou251,000,000125,000262,000,000250,000274,000,000500,000288,000,000
31Imagining Real-Time PCR Measuring Quantities What if YOU started with EIGHT times LESS DNA template than I did?You’d only have 125,000 copies right now at cycle 25…And you’d reach 1,000,000 copies exactly THREE cycles later than I would!
32Imagining Real-Time PCR Measuring Quantities We can easily see that the left-right shift in the curves is related to the starting quantity of DNA!Cq (“Cycle Quantity) values identify the curve positions, based on where they cross a threshold.DNA Quantity and Cq value are related as:Quantity ~ 2CqImagining Real-Time PCR Measuring Quantities252328
33Imagining Real-Time PCR Measuring Quantities We can plot the Cq value versus the Log Quantity on a graph…0.125 unitCq=281 unitCq=254 unitsCq=23… and calculate the quantity of any ‘unknown’ right off of the line!!
34Real-Time PCR Sensitivity How sensitive is Real-Time PCR?Ultimately, even a single copy can be measured! In reality, typically about 100 copies is around the minimum amount.One hundred copies of a 200-bp gene is:twenty attograms (2 x g) of DNA!this is just 2/100ths of a microliter of blood!Real-Time PCR Sensitivity
36How do We Measure DNA in a PCR Reaction? We use reagents that fluoresce in the presence of amplified DNA!5’3’l
37Measuring DNA: Ethidium Bromide + = common and well known- = toxic, not very brightMeasuring DNA: Ethidium Bromide
38Measuring DNA: SYBR Green I + = Bright fluorescence!+ = Low toxicity!Measuring DNA: SYBR Green IAmes test results from Molecular ProbesSinger et al., Mutat. Res. 1999, 439:
39Fluorescent Dyes in PCR Intercalating Dyes SYBR Green in Action!5’3’TaqlPCR makes more double-stranded DNASYBR Green dyebinds to dsDNAWhen illuminated with light at 490nm, the SYBR+DNA complex fluoresces at 520nm.
40Fluorescent Dyes in PCR Other Options Even more ways to detect PCR products:Other Intercalating Dyes- Eva GreenProbes- TaqMan ProbesPrimer/Probe Combinations- Scorpions- LUX Primers
41What Type of Instruments are used with Real-Time PCR? What about the Instruments?Real-time PCR systems consist of THREE main components:Thermal Cycler (PCR machine), linked to a…Optical Module (to detect fluorescence in the tubes during the run), linked to a…Computer (to translate the fluorescence data into meaningful results).
42What Type of Instruments are used with Real-Time PCR? A good example is the MiniOpticon real-time instrument.What Type of Instruments are used with Real-Time PCR?Optical ModuleThermal Cycler Base
43What Type of Instruments are used with Real-Time PCR? One more example is the Bio-Rad CFX-Touch real-time PCR instrument.What Type of Instruments are used with Real-Time PCR?Optical ModuleThermal Cycler BaseThe CFX module scans the PCR plate with LEDs and records fluorescence in each well at each PCR cycle.
44What Type of Software is used with Real-Time PCR? The computer, running real-time software, converts the fluorescent signals in each well to meaningful data.Workflow:Set up PCR protocol.Set up plate layout.Collect data.Analyze data.What Type of Software is used with Real-Time PCR?123,4
45Part 4: What does actual real-time data look like, and what are melt curves?
46Real-Time PCR Actual Data This is some actual data from a recent real-time PCR run.Data like this can easily be generated by preparing a dilution series of DNA.Real-Time PCR Actual Datac366939
47Real-Time PCR Final Product The final product of real-time PCR is a table of Ct values, from which amounts of DNA can be determined.WellFluorContentCycle Quantity( Cq )A03SYBRStd-18.90A04Std-212.20A05Std-315.34A06Std-418.77A07Std-521.84A08Std-625.24A09Std-728.82B038.85B0412.12B0515.31B0618.69B0721.76B08
48Real-Time PCR Melt Curves The fluorescence data collected during PCR tells us “how much” ……. but there is another type of analysis we can do that tells us “what”!Real-Time PCR Melt Curvesc366939
49Melt curves can tell us what products are in a reaction. The principle of melt curves is that as DNA melts (becomes single stranded), DNA-binding dyes will no longer bind and fluoresce.Melt Curves Basics5’3’IDCOLD5’3’IDMEDIUM5’3’HOT
50Melt Curves BasicsMelt curves can tell us what products are in a reaction.PCR products that are shorter or lower G+C will melt at lower temperatures.Different PCR products will therefore have different shaped curves.5’3’IDRFU vs Temp
51Melt Curves Typical Data For convenience, we typically view the derivative (slope) of the actual melt curve data.The resulting graph looks like a chromatogram, with peaks that represent different PCR products.Melt Curves Typical DatadRFU/dTSlope vs. TempTeaching Tip:Use Melt Curves to bring up a good discussion of why different DNA sequences will “melt” at different temperatures! Talk about base-pairing, secondary structure, energy levels, etc!
52Melt Curves High-Resolution Analysis The new field of Precision Melt Analysis even allows differentiation between PCR products based on a single-base pair mismatch!PMA/HRM is now used in mutation screening, detection of biological diversity, and genetic analysis.Melt Curves High-Resolution AnalysisPCR melt data from different organisms is first collected….Then normalized….Then the organisms are compared against each other.
53Part 5: How can we demonstrate real-time PCR in the classroom?
54Crime Scene Investigator Kit in Real-Time CSI: MATERIALS REQUIREDTo run the CSI Kit in real-time, only a few additional items are needed…iQ SYBR Green Supermixand appropriate tubes/capsA real-time PCR instrument
55Crime Scene Investigator Kit in Real-Time CSI: INSTRUCTIONSAn Application Note and a Starter Kit are available ( EDU):The PCR reactions in the CSI kit can be run in real-time as an add-on to the regular kit.
56Crime Scene Investigator Kit in Real-Time CSI: FINAL RESULTSThe Crime Scene Real-Time Extension is great for first-time users!!Teach PCR, Real-Time, Melt Curves, etc!
57GMO Investigator Kit in Real-Time GMO: MATERIALS REQUIREDTo run the GMO Kit in real-time, only two additional items are needed…iQ SYBR Green Supermixand appropriate tubes/capsA real-time PCR instrument
58GMO Investigator Kit in Real-Time GMO: INSTRUCTIONSAn Application Note and Starter Kit ( EDU) are available:Basically, run GMO kit with real-time reagents on a real-time instrument.GMO Investigator Kit in Real-Time
59GMO Investigator Kit in Real-Time GMO: FINAL PRODUCTUltimately, you can even calculate the percentage GMO content in actual food samples!The GMO Real-Time extension is perfect for more advanced users!FoodPlant CqGMO CqDeltaCqPlantGMOPlant ddCqRatio2ddCt%100% GMO23271100Non-GMO2439-1-12-110.00040.04Product A2230-3-4.06256.3Product B2529-2
60Real-Time in the Classroom Getting Started Getting started with teaching Real-Time PCR in the classroom is easy !Use the Crime Scene Real-Time starter kit.Use the GMO Investigator Real-Time kit.Use your own PCR primers and templates with SYBR Green Supermix.It’s nothing more complicated than using a slightly different polymerase supermix, correct plastics, and running on a real-time instrument !
61Why Real-Time in the Classroom is Cool! Real-Time PCR in the classroom is “cool” for a number of reasons!Instant Results! You can see amplification within minutes of starting the PCR run. No waiting until next lab period and waiting for gels to run.What-Will-Happen-Next Factor! Because samples amplify at different times, many students will want to wait for “their” sample to amplify so they can see what happens!Demystifying the Black Box! Now students can see what happens inside the PCR machine!Finally a way to connect Computers and Biology! Many “computery” students get really excited to see the computer control of the instrument and data collection / analysis!
62Conclusions We’ve covered the following topics today: What is real-time PCR used for?How does real-time PCR work?What chemicals and instruments are used to detect DNA?What does real-time data look like?How can we demonstrate real-time PCR in the classroom?
63Resources and References David PalmerBio-Rad Technical Support1(800)4BIORADconsult.bio-rad.comBio-Rad Explorer website:Bio-Rad ExplorerCrime Scene Investigator PCR Basics Kit Real-Time PCR Application NoteBulletinGMO Investigator Kit Real-Time PCR Application NoteBulletinReal-Time PCR Applications GuideBulletin 5279Resources and References
65Today’s Experiment: An Overview Today we’ll use the DNA in the Crime Scene Kit to make some dilutions for our real-time experiment!Each workgroup will have DNA from the Crime Scene kit that has been diluted 1:10, 1:100, 1:1000, 1:10000, or undiluted. This is your “Unknown” DNA.Each workgroup will prepare four real-time PCR reactions:Unknown DNA (replicate 1)Unknown DNA (replicate 2)Unknown DNA diluted 1:100 (replicate 1)Unknown DNA diluted 1:100 (replicate 2)If all goes well, you’ll be able to tell from the Cq values:Which unknown DNA you started with,How accurate your pipetting is,Whether your mini-dilution series demonstrates high-efficiency PCR.
66Today’s Experiment: Step-By-Step Step 1: DNA Dilutions Dilute your “Unknown” DNA 1:100.Mix 1 ul of your DNA (screw-cap tube labelled 1-5) into 99 ul of water (screw-cap tube labelled “W”).Step 2: Prepare your PCR TubesAdd 20 ul of the spiked SYBR Green Supermix (from the screw-cap) tube labelled SMX to four PCR tubes.Step 3: Add DNA to your PCR TubesAdd 20 ul of your DNA samples to each PCR tube:Unknown Replicate AUnknown Replicate BUnknown 1:100 Replicate AUnknown 1:100 Replicate BMix gently, avoiding bubbles!Label appropriately.Step 4: Cap and Load the PCR TubesPlace the optically-clear flat caps on the tubes.Place your reactions in the real-time PCR machine.Today’s Experiment:Step-By-Step
67Today’s Experiment: PCR Protocol Our PCR protocol will look like this: 1. 95C for 3 min (activates Taq)2. 95C for 10 sec (denatures)3. 52C for 30 sec (extend / anneal)4. Plate read (captures fluorescence data)5. Goto Step 2 for 39 more times
68Real-Time PCR David A. Palmer, Ph.D. Technical Support, Bio-Rad LaboratoriesAdjunct Professor, Contra Costa College
69BONUS: How do we optimize Real-Time PCR and troubleshoot problems?
70Optimization of real-time PCR reactions is important: Since real-time PCR calculations are based on a doubling of product every cycle, if the reaction isn’t optimized, this doubling will not occur.OptimizationWhy?
71OptimizationExampleA well-optimized reaction will have evenly spaced standard curves with tight replicates:At 100% efficiency, 10-fold serial dilutions will be spaced 3.3 cycles apart from each other.
72Optimization Basics Optimization is normally done as follows: Design multiple primer sets.Empirically test each primer set with a standard curve.Select best primer set, then run a temperature gradient experiment to determine best annealing temperature.Standard curves are ideal for assessing optimization.OptimizationBasics
73Trouble-shooting Skills Why?Why is it important for teachers to be able to solve real-time PCR problems?Help students have better success with their projects!Preventing problems at the start can help avoid lost experiments and reagents!Being able to explain unusual results leads to great teaching opportunities!Teaching Tip:Very often a student’s first real-time data isn’t perfect – this makes for a great chance to teach better pipetting skillls, experimental design, etc!
74A successful real-time PCR experiment will have the following characteristics: Trouble-ShootingCurves are all S-shapedPlateau height doesn’t matterDilution series has expected spacingCurves are smoothReplicates are tightly clusteredBaselines are relatively flatMelt curve has one peak per product.
75Trouble-Shooting Replicates Replicates are tightly clusteredReplicates are not tightly clusteredIf replicates aren’t tightly clustered, suspect:Pipetting errorPoorly optimized PCR reactionsSample evaporationUnknowns outside of range of detectionInstrument calibration
76Trouble-Shooting Baselines Baseline not flatBaselines are relatively flatIf baselines aren’t flat, suspect:Sample evaporationBubblesReagents not thoroughly mixedBaseline “window” not properly set
77Trouble-Shooting Dilutions Dilution series has expected spacingDilution series tightly compressedTrouble-Shooting Dilutions1000100101Dilution series stretched outIf the dilution series comes out “compressed” or “stretched”, suspect:PipettingToo much DNA (for your assay)PCR inhibitorsToo little DNA (for your assay)Poor PCR efficiency
78Trouble-Shooting Curve Shape Curves are all S-shapedTrouble-Shooting Curve ShapeCurves are not S-shapedIf curves are not S-shaped, suspect:Curves are not actual PCR products!Sample evaporationFluorescence drift in unamplified samplesSomething seriously wrong with assay
79Trouble-Shooting Curve Shape Curves are not smoothCurves are smoothIf curves are not smooth, suspect:Poor pipetting (bubbles)Sample evaporationPoor assay (low fluorescence reagents)System malfunction (line noise)
80Trouble-Shooting Melt Peaks Melt curve has one peak per product.Melt curves have multiple peaks.If melt curves have more than one peak:More than one productPossible normal primer-dimersUsing too low an annealing temperaturePrimers need to be redesigned
81? Trouble-Shooting Common themes in troubleshooting: Care in pipetting.Care in choice of plastics and sealing the plates.Care in experimental design.Use of Positive and Negative Controls.?Teaching Tip:Use Real-Time PCR to teach the importance of properly designed experiments !!
82Real-Time PCR David A. Palmer, Ph.D. Technical Support, Bio-Rad LaboratoriesAdjunct Professor, Contra Costa College