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DNA
BRCA1
mRNA
Protein
Real-Time Example: HIV Treatment
PCR in
Disease Drug treatment for HIV infection often depends on
Management monitoring the “viral load”.
Real-Time PCR allows for direct measurement of the
amount of the virus RNA in the patient.
Virus
RNA
Real-Time Example: Determining percentage of GMO food
PCR in Food content
Seed
wt DNA
GMO DNA
Part 2:
How does Real-Time PCR work?
How does To best understand what real-time PCR
real-time is, let’s review how regular PCR
PCR work? works...
The 3’ 5’
Polymerase
5’ 3’
Chain
d.NTPs
Primers
Reaction
Thermal Stable
DNA Polymerase
Denaturation
Annealing
The 3’ 5’
Polymerase
Chain
5’ 3’
Reaction Extension
PCR work?? 5’
Taq 3’
Extension Continued
3’ Taq 5’
Taq 3’
Repeat
The 3’
3’
5’
Polymerase 3’ 5’
3’
Cycle 2
Chain 3’ 5’
4 Copies
Reaction
3’
3’
3’
How does
PCR work?? 3’
3’
3’
5’
5’
3’
3’ 5’
3’
3’
5’
Cycle 3
3’
3’ 5’
3’ 8 Copies
3’ 5’
3’
3’ 5’
3’
3’
3’
How does …So that’s how traditional PCR is usually presented.
Real-Time
PCR work? In order to understand real-time PCR, let’s use a
“thought experiment”, and save all of the
calculations and formulas until later…
NO GRAPHS
(yet)
Imagining To understand real-time PCR, let’s imagine
Real-Time ourselves in a PCR reaction tube at cycle
PCR number 25…
Imagining
Real-Time
PCR
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
0 5 10 15 20 25 30 35 40
Imagining
Real-Time
PCR ?
How did we So, right now we’re at cycle 25 in a soup with
get here? 1,000,000 copies of the target.
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
0 5 10 15 20 25 30 35 40
Imagining
Real-Time
PCR
So where
What’s it going to be like after the next cycle, in cycle 26?
Probably there will be 2,000,000 amplicons.
are we And cycle 27?
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
Imagining
Real-Time How can all this be used to measure DNA
PCR quantities??
5000000
Measuring
4500000
4000000
Quantities 3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
Imagining Let’s imagine that you start with four times as
Real-Time much DNA as I do…picture our two tubes at
PCR cycle 25 and work backwards a few cycles.
Measuring Cycle 25
Quantities
Cycle Me You
23 250,000 1,000,000
24 500,000 2,000,000
25 1,000,000 4,000,000
Imagining So, if YOU started with FOUR times as much
Real-Time DNA template as I did…
PCR …Then you’d reach 1,000,000 copies exactly
TWO cycles earlier than I would!
Measuring 5000000
Quantities 4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
Imagining What if YOU started with EIGHT times LESS
Real-Time DNA template than I did?
PCR
Cycle 25
Measuring
Quantities 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
What if YOU started with EIGHT times LESS DNA template
Imagining than I did?
Real-Time
PCR You’d only have 125,000 copies right now at cycle 25…
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
We describe the position of the lines with a value that
Imagining represents the cycle number where the trace crosses an
Real-Time arbitrary threshold.
Quantities 5000000
4500000
4000000
Ct Values:
3500000
3000000
2500000
23 25 28
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
Let’s recap…
Imagining
Real-Time
PCR
Measuring 4 units
Ct=23
1 unit
Ct=25
1/8 unit
Ct=28
Quantities 5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
Imagining There’s a DIRECT relationship between the
Real-Time
starting amount of DNA, and the cycle
number that you’ll reach an arbitrary number
PCR of DNA copies (Ct value).
35
30
y = -3 . 3 1 9 2 x + 3 9 .77 2
2
R = 0 .9 9 6 7
25
Ct
20
15
10
0
0 1 2 3 4 5 6 7 8 9 10 11
n
L o g o f c o p y n u m b e r (1 0 )
Imagining How sensitive is Real-Time PCR?
Real-Time
PCR
40
Copy Number vs. Ct - Standard Curve
35
30
y = -3.3192x + 39.772
2
25
R = 0.9967
Ct
20
Measuring
15
10
Quantities
5
0
0 1 2 3 4 5 6 7 8 9 10 11
http://www.web.virginia.edu/Heidi/chapter12/chp12.htm
Measuring SYBR Green I
DNA: SYBR
Green I
Dyes in PCR 5’ 3’
Extension
Intercalating
Dyes Taq 5’
3’
ID ID
5’ ID ID ID 3’
Taq
Apply Excitation
Wavelength
l l l
3’
ID Taq 5’
ID ID ID ID
ID ID ID ID
Taq 3’
l
l
Repeat
Fluorescent Q
Dyes in PCR
R
5’ 3’
Extension
Probes Q
R
Taq
5’ 3’
R
Q
Hydrolysis
Taq
5’
5’ 3’
R
Taq
5’
5’ 3’
l
R
Signal
Taq
5’
5’ 3’
What Type Real-time PCR instruments consist of THREE
of main components:
Instruments
are used 1. Thermal Cycler (PCR machine)
with Real-
Time PCR? 2. Optical Module (to detect fluorescence in
the tubes during the run)
Instruments
are used
with Real-
Time PCR?
What Type Another example is the MiniOpticon real-
of time instrument.
Instruments
are used
with Real-
Time PCR?
What Type The real-time software converts the
of Software fluorescent signals in each well to
is used with
meaningful data.
Real-Time
PCR? 1.
2.
Set up PCR protocol.
Set up plate layout.
3. Collect data.
4. Analyze data.
1 2 3,4
Part 4:
What does real-time data look like?
• This is some actual data from a recent real-
Real-Time time PCR run.
PCR • Data like this can easily be generated by
preparing a dilution series of DNA.
Actual Data
c366939
• The same data set in log view
Real-Time
PCR
Actual Data
• Once threshold is set, Ct values can be
Real-Time calculated automatically by software.
PCR
Setting
Thresholds
Actual Data
c366939
• Melt curves can tell us what products are
Real-Time in a reaction.
• Based on the principle that as DNA melts
PCR – the (becomes single stranded), intercalating
Concept of dyes will no longer bind and fluoresce.
MELT
CURVES…
COLD
3’ 5’
5’ ID ID ID 3’
5’
MEDIUM
3’
5’ ID
3’
3’ 5’
HOT
5’ 3’
• Melt curves can tell us what products are
Real-Time in a reaction.
PCR – the
Concept of
MELT
CURVES… RFU vs T
dRFU/dT
• Different amplicons will have different melt
Real-Time peaks.
PCR • Primer-Dimers will have a very different
melt peak.
The Concept
of MELT
CURVES
STR region
Example of The TH01 locus contains repeats of TCAT.
an STR
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
DAD’S CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT
TCAT TCAT TCAT TCAT AAA
To visualize
TH01 Allele Mother Father Child C Child D Child E
alleles ladder
PCR products
Crime Scene (14)
investigators (13)
electrophoresis (11)
(10)
(9)
(8)
(7)
(6)
(5)
(4)
(3)
Real STR
analysis
Four different
fluorescent tags
have been used
to identify 7
amplified loci
Use a 3% gel to
separate small
fragment sizes Gel running
Analysis of
Results:
AL CS A B C D
Who can’t be
excluded?
15
BXP007 alleles
10 AL: Allele ladder
7 CS: Crime Scene
5 A: Suspect A
4 B: Suspect B
C: Suspect C
3 D: Suspect D
2
1
genotype
Core Content
(Crime Scene
Kit)
Crime Scene
Investigator
Kit So how can we use
the Crime Scene Kit to
perform real-time
PCR???
Two options…
Crime Scene
Investigator • Introduction to DNA profiling
PCR Basics
Kit in REAL- • Set up PCR reactions on a real-time
TIME! PCR instrument, using real-time
reagents
Option 1
• Electrophorese PCR products
Option 2
dRFU/dT
Crime Scene • Learning Points
Investigator – Viewing PCR reactions as they occur in real-time
• Exciting!
PCR Basics – Using real-time PCR to quantify DNA
Kit in REAL- • Basis of gene expression analysis, disease
TIME! diagnosis, etc.
– Measuring pipetting variation
• Run samples in duplicate for an easy test of
Option 2 reproducibility
– Importance of experimental controls
• No template control and positive controls
– Melt curve analysis
• Tie concepts of the basic structure of DNA with
visible evidence that two strands can anneal and
melt.
Crime Scene •To run either of the two options,
Investigator ONLY two additional items are
Kit in Real- needed!
Time !
•iQ SYBR Green Supermix
• Each workgroup will have DNA from the Crime Scene kit
that has been diluted 1:10, 1:100, 1:1000, 1:10000, or
undiluted.
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