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1. 2 David A. Palmer, Ph.D. Technical Support, Bio-Rad Laboratories Adjunct Professor, Contra Costa College Real-Time PCR.

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Presentation on theme: "1. 2 David A. Palmer, Ph.D. Technical Support, Bio-Rad Laboratories Adjunct Professor, Contra Costa College Real-Time PCR."— Presentation transcript:

1 1

2 2 David A. Palmer, Ph.D. Technical Support, Bio-Rad Laboratories Adjunct Professor, Contra Costa College Real-Time PCR

3 3 Objectives Today we’ll talk about Real-Time PCR: 1.What is real-time PCR used for? 2.How does real-time PCR work? 3.What chemicals and instruments are used to detect DNA? 4.What does real-time data look like? 5.How can we demonstrate real-time PCR in the classroom?

4 4 Part 1: What is Real-Time PCR and what is it used for?

5 5 What 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!

6 6 What is Real-Time PCR? Conventional PCR tells us “what”. Real-Time PCR tells us “how much”.

7 7 What is Real-Time PCR used for? Real-Time PCR has become a cornerstone of molecular biology. Just some of the uses include: Gene expression analysis –Cancer and Drug research Disease diagnosis and management –Viral quantification Food testing –Percent GMO food Animal and plant breeding –Gene copy number Forensics –Sample identification and quantification

8 8 Real-Time PCR in Gene Expression Analysis Example: BRCA1 Expression Profiling BRCA1 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. DNA mRNA Protein BRCA1 Real-Time PCR Determine gene expression and publish scientific paper!

9 9 Real-Time PCR in Disease Management Example: HIV Treatment Drug 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. Viral RNA Measure amount of virus, adjust prescriptions. Real-Time PCR

10 10 Real-Time PCR in Food Testing Example: Determining percentage of GMO food content Determination of percent GMO food content important for import / export regulations. Labs use Real-Time PCR to measure amount of transgenic versus wild-type DNA. wt DNA GMO DNA Real-Time PCR International shipments depend on results!

11 11 Real-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 ??

12 12 Part 2: How does Real-Time PCR work?

13 13 How does real-time PCR work? To best understand what real-time PCR is, let’s review how regular PCR works...

14 14 The Polymerase Chain Reaction How does PCR work?? 5’ 3’ d. NTPs Thermal Stable DNA Polymerase Primers 5’ 3’ 5’3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’3’ 5’3’ 5’3’ Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’3’ 5’3’ Annealing Add to Reaction Tube

15 15 The Polymerase Chain Reaction How does PCR work?? Extension 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Extension Continued 5’ 3’ 5’ 3’ 5’ Taq 3’ 5’ 3’ Taq 5’ Repeat

16 16 The Polymerase Chain Reaction How does PCR work?? 5’3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’3’ 5’ 3’ Cycle 2 4 Copies Cycle 3 8 Copies 5’ 3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’3’ 5’ 3’

17 17 How 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…

18 18 Imagining Real-Time PCR To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…

19 19 Imagining 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.

20 20 Imagining 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.

21 21 Imagining 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:

22 22 Imagining 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? ?

23 23 Imagining 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.

24 24 Imagining 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 1,000,000,000,000,000,000,000,000,000,000,000,000,0 00,000,000,000,000,000,000 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!!!!

25 25 Imagining 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!

26 26 Imagining 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:

27 27 Imagining Real-Time PCR Measuring Quantities How can all this be used to measure DNA quantities??

28 28 Imagining 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. CycleMeYou 251,000,0004,000, ,0002,000, ,0001,000,000 Cycle 25

29 29 Imagining 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!

30 30 Imagining Real-Time PCR Measuring Quantities What if YOU started with EIGHT times LESS DNA template than I did? CycleMeYou 251,000,000125, ,000,000250, ,000,000500, ,000,0001,000,000 Cycle 25

31 31 Imagining 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!

32 32 Imagining 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 ~ 2 Cq

33 33 Imagining Real-Time PCR Measuring Quantities We can plot the Cq value versus the Log Quantity on a graph… 4 units Cq=23 1 unit Cq= unit Cq=28 … and calculate the quantity of any ‘unknown’ right off of the line!!

34 34 Real-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 2/100 ths of a microliter of blood!

35 35 Part 3: How do we detect and measure DNA?

36 36 How do We Measure DNA in a PCR Reaction? We use reagents that fluoresce in the presence of amplified DNA! 5’ 3’ 5’

37 37 Measuring DNA: Ethidium Bromide Ethidium Bromide + = common and well known - = toxic, not very bright

38 38 Measuring DNA: SYBR Green I SYBR Green I + = Bright fluorescence! + = Low toxicity! Ames test results from Molecular Probes Singer et al., Mutat. Res. 1999, 439:

39 39 Fluorescent Dyes in PCR Intercalating Dyes PCR makes more double-stranded DNA SYBR Green dye binds to dsDNA When illuminated with light at 490nm, the SYBR+DNA complex fluoresces at 520nm. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Taq 3’ 5’ 3’ Taq 5’ SYBR Green in Action!

40 40 Fluorescent Dyes in PCR Other Options Even more ways to detect PCR products: Other Intercalating Dyes - Eva Green Probes - TaqMan Probes Primer/Probe Combinations - Scorpions - LUX Primers

41 41 What Type of Instruments are used with Real-Time PCR? What about the Instruments? Real-time PCR systems consist of THREE main components: 1.Thermal Cycler (PCR machine), linked to a… 2.Optical Module (to detect fluorescence in the tubes during the run), linked to a… 3.Computer (to translate the fluorescence data into meaningful results).

42 42 What Type of Instruments are used with Real-Time PCR? A good example is the MiniOpticon real-time instrument. Optical Module Thermal Cycler Base

43 43 What Type of Instruments are used with Real- Time PCR? The CFX module scans the PCR plate with LEDs and records fluorescence in each well at each PCR cycle. One more example is the Bio-Rad CFX-Touch real-time PCR instrument. Optical Module Thermal Cycler Base

44 44 What 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: 1.Set up PCR protocol. 2.Set up plate layout. 3.Collect data. 4.Analyze data. 1 23,4

45 45 Part 4: What does actual real-time data look like, and what are melt curves?

46 46 Real-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. c366939

47 47 Real-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. WellFluorContent Cycle Quantity ( Cq ) A03SYBRStd A04SYBRStd A05SYBRStd A06SYBRStd A07SYBRStd A08SYBRStd A09SYBRStd B03SYBRStd B04SYBRStd B05SYBRStd B06SYBRStd B07SYBRStd B08SYBRStd

48 48 Real-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”! c366939

49 49 Melt Curves Basics Melt 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. 5’ 3’ ID COLD 5’ 3’ ID MEDIUM 5’ 3’ HOT

50 50 Melt Curves Basics RFU vs Temp 5’ 3’ 5’ 3’ ID 5’ 3’ ID Melt 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.

51 51 Melt 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. dRFU/dT Slope vs. Temp Teaching 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!

52 52 Melt 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. PCR melt data from different organisms is first collected…. Then normalized…. Then the organisms are compared against each other.

53 53 Part 5: How can we demonstrate real-time PCR in the classroom?

54 54 CSI: MATERIALS REQUIRED To run the CSI Kit in real-time, only a few additional items are needed… iQ SYBR Green Supermix and appropriate tubes/caps A real-time PCR instrument Crime Scene Investigator Kit in Real-Time

55 55 Crime Scene Investigator Kit in Real-Time CSI: INSTRUCTIONS An 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.

56 56 Crime Scene Investigator Kit in Real-Time CSI: FINAL RESULTS The Crime Scene Real-Time Extension is great for first-time users!! Teach PCR, Real-Time, Melt Curves, etc!

57 57 GMO: MATERIALS REQUIRED To run the GMO Kit in real-time, only two additional items are needed… iQ SYBR Green Supermix and appropriate tubes/caps A real-time PCR instrument GMO Investigator Kit in Real-Time

58 58 GMO Investigator Kit in Real-Time GMO: INSTRUCTIONS An Application Note and Starter Kit ( EDU) are available: Basically, run GMO kit with real-time reagents on a real-time instrument.

59 59 GMO Investigator Kit in Real-Time GMO: FINAL PRODUCT Ultimately, you can even calculate the percentage GMO content in actual food samples! The GMO Real-Time extension is perfect for more advanced users! Food Plant Cq GMO Cq Delta Cq Plant Delta Cq GMO Plant ddCq Ratio 2 ddCt Ratio % 100% GMO Non- GMO Product A Product B

60 60 Getting started with teaching Real-Time PCR in the classroom is easy ! 1.Use the Crime Scene Real-Time starter kit. 2.Use the GMO Investigator Real-Time kit. 3.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 ! Real-Time in the Classroom Getting Started

61 61 Why 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!

62 62 Conclusions We’ve covered the following topics today: 1.What is real-time PCR used for? 2.How does real-time PCR work? 3.What chemicals and instruments are used to detect DNA? 4.What does real-time data look like? 5.How can we demonstrate real-time PCR in the classroom?

63 63 Resources and References David Palmer Bio-Rad Technical Support 1(800)4BIORAD consult.bio-rad.com Bio-Rad Explorer website: Bio-Rad Explorer Crime Scene Investigator PCR Basics Kit Real-Time PCR Application Note Bulletin GMO Investigator Kit Real-Time PCR Application Note Bulletin Real-Time PCR Applications Guide Bulletin 5279

64 64 Real-Time PCR Practical Exercise!

65 65 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. Today’s Experiment: An Overview

66 66 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 Tubes –Add 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 Tubes –Add 20 ul of your DNA samples to each PCR tube: Unknown Replicate A Unknown Replicate B Unknown 1:100 Replicate A Unknown 1:100 Replicate B –Mix gently, avoiding bubbles! –Label appropriately. Step 4: Cap and Load the PCR Tubes –Place the optically-clear flat caps on the tubes. –Place your reactions in the real-time PCR machine. Today’s Experiment: Step-By-Step

67 67 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 Today’s Experiment: PCR Protocol

68 68 David A. Palmer, Ph.D. Technical Support, Bio-Rad Laboratories Adjunct Professor, Contra Costa College Real-Time PCR

69 69 BONUS: How do we optimize Real- Time PCR and troubleshoot problems?

70 70 Optimization 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. Optimization Why?

71 71 A 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. Optimization Example

72 72 Optimization is normally done as follows: 1.Design multiple primer sets. 2.Empirically test each primer set with a standard curve. 3.Select best primer set, then run a temperature gradient experiment to determine best annealing temperature. 4.Standard curves are ideal for assessing optimization. Optimization Basics

73 73 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! Trouble- shooting Skills Why? 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!

74 74 Trouble- Shooting A successful real-time PCR experiment will have the following characteristics: Replicates are tightly clustered Baselines are relatively flat Dilution series has expected spacing Plateau height doesn’t matter Melt curve has one peak per product. Curves are all S-shaped Curves are smooth

75 75 Trouble- Shooting Replicates If replicates aren’t tightly clustered, suspect: –Pipetting error –Poorly optimized PCR reactions –Sample evaporation –Unknowns outside of range of detection –Instrument calibration Replicates are tightly clustered Replicates are not tightly clustered

76 76 Trouble- Shooting Baselines If baselines aren’t flat, suspect: –Sample evaporation –Bubbles –Reagents not thoroughly mixed –Baseline “window” not properly set Baselines are relatively flat Baseline not flat

77 77 Trouble- Shooting Dilutions Dilution series has expected spacing Dilution series tightly compressed Dilution series stretched out If the dilution series comes out “compressed” or “stretched”, suspect: –Pipetting –Too much DNA (for your assay) –PCR inhibitors –Too little DNA (for your assay) –Poor PCR efficiency

78 78 Trouble- Shooting Curve Shape If curves are not S-shaped, suspect: –Curves are not actual PCR products! –Sample evaporation –Fluorescence drift in unamplified samples –Something seriously wrong with assay Curves are all S-shaped Curves are not S-shaped

79 79 Trouble- Shooting Curve Shape If curves are not smooth, suspect: –Poor pipetting (bubbles) –Sample evaporation –Poor assay (low fluorescence reagents) –System malfunction (line noise) Curves are smooth Curves are not smooth

80 80 Trouble- Shooting Melt Peaks If melt curves have more than one peak: –More than one product –Possible normal primer-dimers –Using too low an annealing temperature –Primers need to be redesigned Melt curve has one peak per product. Melt curves have multiple peaks.

81 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 !!

82 82 David A. Palmer, Ph.D. Technical Support, Bio-Rad Laboratories Adjunct Professor, Contra Costa College Real-Time PCR


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