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

3. Objectives 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?

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

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

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. 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.
Determine gene expression and publish scientific paper!
Real-Time PCR
DNA
BRCA1
mRNA
Protein

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.
Real-Time PCR
Viral RNA
Measure amount of virus, adjust prescriptions.

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.
Real-Time PCR
wt DNA
GMO DNA
International shipments depend on results!

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

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

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

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

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

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

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. 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. 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. 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. 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. 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 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 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!!!!

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

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.
Cycle 25
Cycle Me
You 25
1,000,000
4,000,000 24
500,000
2,000,000 23
250,000

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!
Imagining Real-Time PCR Measuring Quantities

30. Imagining Real-Time PCR Measuring Quantities
What if YOU started with EIGHT times LESS DNA template than I did?
Cycle 25
Cycle Me
You 25
1,000,000
125,000 26
2,000,000
250,000 27
4,000,000
500,000 28
8,000,000

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. 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 ~ 2Cq
Imagining Real-Time PCR Measuring Quantities 25 23 28

33. Imagining Real-Time PCR Measuring Quantities
We can plot the Cq value versus the Log Quantity on a graph…
0.125 unit
Cq=28
1 unit
Cq=25
4 units
Cq=23
… and calculate the quantity of any ‘unknown’ right off of the line!!

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 2/100ths of a microliter of blood!
Real-Time PCR Sensitivity

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

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

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

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

39. Fluorescent Dyes in PCR Intercalating Dyes
SYBR Green in Action! 5’ 3’
Taq l
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.

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. What 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).

42. What 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 Module
Thermal Cycler Base

43. What 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 Module
Thermal Cycler Base
The CFX module scans the PCR plate with LEDs and records fluorescence in each well at each PCR cycle.

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:
Set up PCR protocol.
Set up plate layout.
Collect data.
Analyze data.
What Type of Software is used with Real-Time PCR? 1 2
3,4

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

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.
Real-Time PCR Actual Data
c366939

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.
Well
Fluor
Content
Cycle Quantity
( Cq )
A03
SYBR
Std-1
8.90
A04
Std-2
12.20
A05
Std-3
15.34
A06
Std-4
18.77
A07
Std-5
21.84
A08
Std-6
25.24
A09
Std-7
28.82
B03
8.85
B04
12.12
B05
15.31
B06
18.69
B07
21.76
B08

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”!
Real-Time PCR Melt Curves
c366939

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

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

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.
Melt Curves Typical Data
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. 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.
Melt Curves High-Resolution Analysis
PCR melt data from different organisms is first collected….
Then normalized….
Then the organisms are compared against each other.

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

54. Crime Scene Investigator Kit in Real-Time
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

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. 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. GMO Investigator Kit in Real-Time
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

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.
GMO Investigator Kit in Real-Time

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
GMO
Plant ddCq
Ratio
2ddCt %
100% GMO 23 27 1
100
Non-GMO 24 39 -1
-12
-11
0.0004
0.04
Product A 22 30 -3 -4
.0625
6.3
Product B 25 29 -2

60. Real-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 !

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. Conclusions 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?

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
Resources and References

64. Real-Time PCR Practical Exercise!

65. Today’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.

66. Today’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 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. Today’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

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

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

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. Optimization
Example
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.

72. Optimization 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.
Optimization
Basics

73. Trouble-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!

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

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

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

77. Trouble-Shooting Dilutions
Dilution series has expected spacing
Dilution series tightly compressed
Trouble-Shooting Dilutions
1000
100 10 1
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. Trouble-Shooting Curve Shape
Curves are all S-shaped
Trouble-Shooting Curve Shape
Curves are not S-shaped
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

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

80. Trouble-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 product
Possible normal primer-dimers
Using too low an annealing temperature
Primers 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 !!

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