1. Real-Time PCR

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

3. 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!

4. 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”.

5. What is Real-Time PCR used for?
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

6. 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

7. 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.

8. 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!

9. 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 ??

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

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

12. 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

13. 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

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

15. 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…

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

17. 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.

18. 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.

19. 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:

20. 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?

21. Imagining Real-Time PCR How did we get here?
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.

22. Imagining Real-Time PCR How did we get here?
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!!!!

23. 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!

24. Imagining Real-Time PCR How did we get here?
If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this:

25. Imagining Real-Time PCR How did we get here?
How can all this be used to measure DNA quantities??

26. Imagining Real-Time PCR How did we get here?
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

27. 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!

28. 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

29. 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!

30. 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 25 23 28

31. 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!!

32. 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!

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

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

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

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

37. 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.

38. Fluorescent Dyes in PCR - Other Options
Even more ways to detect PCR products:
Probes
- TaqMan Probes

39. The TaqMan® technique
The probe contains a fluorescence reporter and a quencher.
Important:
The annealing temp. of the probe has to be higher than the annealing temp. of the primers!

40. Fluorescence Resonance Energy Transfer (FRET)
In addition to the 5’ nuclease activity, TaqMan utilizes FRET or energy transfer. The 5’ reporter dye has a higher energy than the 3’ quencher dye. When the probe is intact, energy from the 5’ dye is transferred (energy flows down hill) to the 3’ dye. Thus, the intact probe has a low emission from the 5’ dye and high emission from the 3’ dye.
When the probe is cleaved, the distance between the two free dyes increases to the extent that the energy transfer cannot occur. Thus, the 5’ dye now has a greater emission than the 3’ dye.

41. What Type of Instruments are used with Real-Time PCR?
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. Part 4: What does actual real-time data look like, and what are melt curves?

43. 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.

44. 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

45. 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”!

46. 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

47. 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

48. 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!

49. 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.

50. Homozygote Amplification
One
Homoduplex

51. Observed Combination of 4 Duplexes Heterozygote Amplification Two Two
Homoduplexes
Two
Heteroduplexes

52. Small Amplicon Primer Design
Primers are designed to be as close as possible to the SNP site
The sequence of the primers must be checked for primer-primer dimer formation

53. Engineered SNP pBR322 Plasmids

54. Clinical Samples

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

56. Optimization
Why?
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.

57. 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

58. Optimization is normally done as follows:
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.

59. 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.

60. 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

61. 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

62. 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

63. 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

64. 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)

65. 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

66. ? 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 !!