1
Real-Time PCR
David A. Palmer, Ph.D.
Technical Support, Bio-Rad Laboratories
Adjunct Professor, Contra Costa College
2
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?
3
Part 1:
What is Real-Time PCR
and what is it used for?
4
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!
5
What is
Real-Time
PCR?
Conventional PCR
tells us “what”.
Real-Time PCR
tells us “how much”.
6
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
7
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
BRCA1
mRNA
Protein
8
Determine gene
expression and
publish scientific
paper!
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.
9
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
International shipments
depend on results!
10
Real-Time
PCR in
Forensics
What is it ??
Enough DNA to ID ??
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!
11
Part 2:
How does Real-Time PCR
work?
12
How does
real-time
PCR work?
13
To best understand what real-time PCR
is, let’s review how regular PCR
works...
The
Polymerase
Chain
Reaction
How does
PCR work??
5’
3’
5’
3’
d.NTPs
Primers
Thermal Stable
DNA Polymerase
Add to Reaction Tube
Denaturation
Annealing
14
The
Polymerase
Chain
Reaction
How does
PCR work??
3’
5’
5’
3’
Extension
Taq
3’
5’
5’
3’
Taq
Extension Continued
Taq 5’
3’
Taq
3’
Repeat
15
The
Polymerase
Chain
Reaction
How does
PCR work??
3’
3’
3’
5’
3’
3’
5’
3’
3’
5’
Cycle 2
4 Copies
3’
3’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
3’
5’
3’
5’
Cycle 3
8 Copies
3’
3’
16
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…
17
Imagining
Real-Time
PCR
18
To understand real-time PCR, let’s imagine
ourselves in a PCR reaction tube at cycle
number 25…
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.
19
Imagining
Real-Time
PCR
How did we
get here?
20
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.
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:
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
21
0
5
10
15
20
25
30
35
40
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?
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
22
0
5
10
15
20
25
30
35
40
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.
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
23
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
So where
are we
going?
24
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!!!!
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!
25
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:
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
26
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
How can all this be used to measure
DNA quantities??
5000000
Measuring
Quantities
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
27
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
Measuring
Quantities
28
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
1,000,000
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!
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
29
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
What if YOU started with EIGHT times LESS
DNA template than I did?
Cycle 25
Measuring
Quantities
30
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
1,000,000
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!
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
31
0
5
10
15
20
25
30
35
40
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
5000000
4500000
4000000
3500000
25
23
3000000
2500000
28
2000000
1500000
1000000
500000
0
32
0
5
10
15
20
25
30
35
40
Imagining
Real-Time
PCR
We can plot the Cq value versus the
Log Quantity on a graph…
30
28
27
26
Ct
Measuring
Quantities
29
0.125 unit
Cq=28
25
24
1 unit
Cq=25
23
22
21
20
0.1
1
Log Quantity
4 units
Cq=23
… and calculate the quantity of any
‘unknown’ right off of the line!!
33
10
Real-Time
PCR
How sensitive is Real-Time PCR?
Copy Number vs. Ct - Standard Curve
40
35
30
y = -3.3192x + 39.772
2
Ct
Sensitivity
R = 0.9967
25
20
15
10
5
0
0
1
2
3
4
5
6
7
8
9
10
11
n
Log of copy number (10 )
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 10-17 g) of DNA!
• this is just 2/100ths of a microliter of blood!
34
Part 3:
How do we detect and
measure DNA?
35
How do We
Measure
DNA in a
PCR
Reaction?
We use reagents that fluoresce in the
presence of amplified DNA!
l
3’
36
l
l
5’
Measuring
DNA:
Ethidium
Bromide
37
Ethidium Bromide
+ = common and well known
- = toxic, not very bright
http://www.web.virginia.edu/Heidi/chapter12/chp12.htm
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: 37- 47
38
Fluorescent
Dyes in PCR
Intercalating
Dyes
SYBR Green in Action!
3’
5’
5’
3’
PCR makes more
double-stranded DNA
Taq
3’
5’
5’
3’
Taq
SYBR Green dye
binds to dsDNA
l
l
l
3’
3’
Taq
l
39
Taq5’
l
When illuminated with light at
490nm, the SYBR+DNA complex
fluoresces at 520nm.
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
40
What Type of What about the Instruments?
Instruments
are used with
Real-time PCR systems consist of THREE main
Real-Time
components:
PCR?
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).
41
What Type of
Instruments
are used with
Real-Time
PCR?
42
A good example is the MiniOpticon real-time
instrument.
Optical Module
Thermal Cycler Base
What Type
of
Instruments
are used
with RealTime PCR?
43
One more example is the Bio-Rad CFX-Touch
real-time PCR instrument.
Optical Module
Thermal Cycler Base
The CFX module scans the PCR plate with
LEDs and records fluorescence in each
well at each PCR cycle.
What Type
of Software
is used with
Real-Time
PCR?
1
44
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.
2
3,4
Part 4:
What does actual real-time
data look like, and what
are melt curves?
45
Real-Time
PCR
Actual Data
46
• This is some actual data from a recent realtime PCR run.
• Data like this can easily be generated by
preparing a dilution series of DNA.
c366939
Real-Time
PCR
Final Product
47
•
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
SYBR
Std-2
12.20
A05
SYBR
Std-3
15.34
A06
SYBR
Std-4
18.77
A07
SYBR
Std-5
21.84
A08
SYBR
Std-6
25.24
A09
SYBR
Std-7
28.82
B03
SYBR
Std-1
8.85
B04
SYBR
Std-2
12.12
B05
SYBR
Std-3
15.31
B06
SYBR
Std-4
18.69
B07
SYBR
Std-5
21.76
B08
SYBR
Std-6
25.24
Real-Time
PCR
• The fluorescence data collected during PCR tells
us “how much” …
Melt Curves
…. but there is another type of analysis we can do
that tells us “what”!
48
c366939
3’
5’
ID
ID
ID
5’
3’
5’
3’
5’
ID
3’
49
3’
5’
5’
3’
COLD
• The principle of melt curves is that as DNA
melts (becomes single stranded), DNA-binding
dyes will no longer bind and fluoresce.
MEDIUM
Basics
Melt curves can tell us what products
are in a reaction.
HOT
Melt Curves
Melt Curves
•
Melt curves can tell us what
products are in a reaction.
Basics
•
PCR products that are
shorter or lower G+C will
melt at lower temperatures.
•
Different PCR products will
therefore have different
shaped curves.
3’
5’
3’
5’
3’
5’
ID
ID
ID 5’
3’
5’
ID
3’
5’
3’
RFU vs Temp
50
Melt Curves
•
For convenience, we typically view the derivative
(slope) of the actual melt curve data.
Typical Data
•
The resulting graph looks like a chromatogram, with
peaks that represent different PCR products.
dRFU/dT
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!
51
Melt Curves
HighResolution
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.
52
Part 5:
How can we demonstrate
real-time PCR in the
classroom?
53
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
54
Crime Scene
Investigator
Kit
in Real-Time
55
CSI: INSTRUCTIONS
• An Application Note and a Starter Kit are available
(166-2660EDU):
• The PCR reactions in the CSI kit can be run in realtime as an add-on to the regular kit.
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!
56
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
57
GMO
Investigator
Kit
in Real-Time
58
GMO: INSTRUCTIONS
• An Application Note and Starter Kit (166-2560EDU)
are available:
• Basically, run GMO kit with real-time reagents on a
real-time instrument.
GMO
Investigator
Kit
in Real-Time
GMO: FINAL PRODUCT
• Ultimately, you can even calculate the percentage
GMO content in actual food samples!
Food
Plant
Cq
GMO
Cq
Delta
Cq
Plant
Delta
Cq
GMO
GMO
Plant
ddCq
Ratio
2ddCt
Ratio
%
100%
GMO
23
27
0
0
0
1
100
NonGMO
24
39
-1
-12
-11
0.0004
0.04
Product
A
22
30
1
-3
-4
.0625
6.3
Product
B
25
29
-2
-2
0
1
100
• The GMO Real-Time extension is perfect for more
advanced users!
59
Real-Time in
the
Classroom
Getting
Started
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 !
60
Real-Time PCR in the classroom is
Why Real“cool” for a number of reasons!
Time in the
Classroom is • Instant Results! You can see amplification within
Cool!
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!
61
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?
62
Resources
and
References
• David Palmer
David_Palmer@bio-rad.com
• Bio-Rad Technical Support
1(800)4BIORAD
consult.bio-rad.com
• Bio-Rad Explorer website:
www.explorer.bio-rad.com
• Bio-Rad Explorer email:
biotechnology_explorer@bio-rad.com
• Crime Scene Investigator PCR Basics Kit
Real-Time PCR Application Note
Bulletin 166-2505
• GMO Investigator Kit Real-Time PCR
Application Note
Bulletin 166-2605
• Real-Time PCR Applications Guide
Bulletin 5279
63
Real-Time PCR
Practical Exercise!
64
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 highefficiency PCR.
65
Today’s
Experiment:
Step-By-Step
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:
PCR Protocol
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
Real-Time PCR
David A. Palmer, Ph.D.
Technical Support, Bio-Rad Laboratories
Adjunct Professor, Contra Costa College
68
BONUS:
How do we optimize RealTime PCR and
troubleshoot problems?
69
Optimization
Why?
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
• A well-optimized reaction will have evenly
spaced standard curves with tight
replicates:
Example
• At 100% efficiency, 10-fold serial dilutions
will be spaced 3.3 cycles apart from each
other.
71
Optimization
Basics
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.
Troubleshooting
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 realtime data isn’t perfect – this
makes for a great chance to
teach better pipetting skillls,
experimental design, etc!
73
TroubleShooting
• A successful real-time PCR experiment will
have the following characteristics:
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
74
Melt curve has one
peak per product.
TroubleShooting
Replicates
Replicates are
tightly clustered
Replicates are
not tightly
clustered
•
75
If replicates aren’t tightly clustered,
suspect:
– Pipetting error
– Poorly optimized PCR reactions
– Sample evaporation
– Unknowns outside of range of detection
– Instrument calibration
TroubleShooting
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
76
TroubleShooting
Dilutions
Dilution series has
expected spacing
Dilution series
tightly compressed
Dilution series
stretched out
1000
100
10
1
• 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
77
Curves are all
S-shaped
TroubleShooting
Curves are
not S-shaped
Curve Shape
•
78
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
TroubleShooting
Curves are
not smooth
Curve Shape
Curves are
smooth
•
79
If curves are not smooth, suspect:
– Poor pipetting (bubbles)
– Sample evaporation
– Poor assay (low fluorescence reagents)
– System malfunction (line noise)
TroubleShooting
Melt Peaks
Melt curve has one
peak per product.
Melt curves have
multiple peaks.
•
80
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
TroubleShooting
Common themes in troubleshooting:
?
Teaching Tip:
Use Real-Time PCR to
teach the importance of
properly designed
experiments !!
81
Care in pipetting.
Care in choice of plastics and sealing
the plates.
Care in experimental design.
Use of Positive and Negative Controls.
Real-Time PCR
David A. Palmer, Ph.D.
Technical Support, Bio-Rad Laboratories
Adjunct Professor, Contra Costa College
82