Lab 7: QPCR
Today:
1. Turn in Plasmid DNA report & F1 mapping data
2. Introduction to todays lab
- Review of PCR
- QPCR
3. QPCR exercise
i. Prepare standard curve (Procedure 2)
ii. Setup QPCR reactions (Procedure 3)
**QPCR results will be e-mailed to you within 48 hours**
4. Continue fly work, as needed
5. Due next week: QPCR report (see p. 113 for content)
Bring test substance from home for Ames test
Review:
Analysis of
Restriction Enzyme
Digestions
Example of a gel
1 2 3 4 5 6 7 8
1. Untreated
2. RNAse only
3. DNA ladder
4. BamH1 + RNAse
5. BamH1 + RNAse
6. BamH1 + RNAse
7. BamH1 only
8. BamH1 only
Recombinant Plasmid
~4200 bp (4.2 kb)
BamH1 site
Bacterial Tobacco DNA
plasmid
vector
BamH1 site
supercoiled
Recombinant Plasmid circle
~4200 bp (4.2 kb)
BamH1 site
Bacterial Tobacco DNA
plasmid
vector
BamH1 site
supercoiled
Recombinant Plasmid circle
~4200 bp (4.2 kb)
BamH1 site
Bacterial Tobacco DNA
plasmid
vector
BamH1 site
No BamH1
Size: 3000 bp*
Shape: circle
Topology: supercoiled
Identity: recombinant plasmid
* migrates faster
than its true size
(4200 bp) relative
to DNA ladder
supercoiled
Recombinant Plasmid circle
~4200 bp (4.2 kb)
BamH1 site
Bacterial Tobacco DNA
plasmid
vector
BamH1 site
No BamH1
BamH1
Size: 3000 bp
Size: 3000 bp* Shape: linear
Shape: circle Topology: relaxed
Topology: supercoiled Identity: bacterial plasmid
Identity: recombinant plasmid
+ vector
* migrates faster
than its true size Size: 1200 bp
(4200 bp) relative Shape: linear
to DNA ladder Topology: relaxed
Identity: tobacco DNA
PCR
PCR = Polymerase Chain Reaction
For years, cloning was the only way to amplify a
fragment and thus, was a prerequisite for many other
molecular methods of analysis
The polymerase chain reaction (PCR) was developed
in 1983 and allows DNA fragments to be amplified a
billion-fold in just a few hours
This method can amplify extremely small amounts of
original DNA, even a single molecule
PCR revolutionized molecular biology and is now one
of the most widely used of all molecular techniques
PCR = Polymerase Chain Reaction
The basis of PCR is repeated rounds of replication
catalyzed by DNA polymerase
PCR components:
PCR = Polymerase Chain Reaction
The basis of PCR is repeated rounds of replication
catalyzed by DNA polymerase
PCR components:
1) DNA template (DNA target)
2) Two single-stranded DNA primers complementary to
the target sequence, each with a 3-OH group to which
new nucleotides can be added
3) DNA polymerase = Taq polymerase (stable at high temps)
4) dNTPs (dATP, dTTP, dGTP, dCTP)
5) Magnesium ions and other salts needed for the reaction
to proceed
DNA polymerase
PCR = Polymerase Chain Reaction
A typical PCR reaction include three steps
Step 1: Denaturation- reaction is heated to 94oC to separate the
two strands of the DNA template
5 5 3
3
3 5
dsDNA target 3 5
Step 2: Primer annealing- reaction is cooled to allow the
primers to anneal (renature) to their complementary sequences
on the templates
3 5
5 3
5 3
3 5
PCR = Polymerase Chain Reaction
Step 3: Primer extension- reaction is heated to 72oC and DNA
polymerase catalyzes synthesis of new DNA strands
3 5
5 3
3
5
3 5
3 steps = one cycle
After a single cycle, the amount of target DNA has doubled
(1 double helix has increased to 2 double helices)
PCR = Polymerase Chain Reaction
The whole cycle is repeated many times
Target DNA can be amplified >1-billion fold in 30 cycles
After many cycles, ends of most amplified molecules
defined by 5 ends of forward & reverse primers
Considerations in Choosing PCR Primers
To define sequence to be amplified:
Forward & reverse primer sequences are complementary to ends of
DNA region to be amplified
To maximize reaction efficiency & specificity:
Primers will amplify a small (100-200 bp) sequence
15-30 nucleotides in length
Sequences lack inverted repeats, which minimizes secondary structure
Primers are not complementary to one another, which minimizes
formation of primer dimers Primer dimer
forward primer 5 3 forward primer 5 3
3 5 reverse primer 3 5 reverse primer
Primer annealing step Primer extension step
Primers have similar melting temperatures (Tm)
Tm = temperature at which 50% of the primer will anneal to
the template DNA
PCR Kinetics
3 Phases of Amplification:
1. Exponential
Linear Plateau
- reagents plentiful
- target doubles each cycle
PCR Product (Log 2)
2. Linear
- reagents become limiting
- target does not double each cycle
Exponential - when transition from exponential to
linear occurs can vary between
different reactions
3. Plateau
PCR Cycle Number - reagents depleted
- target levels change little with
additional cycles
End Point PCR
For many applications, PCR products are assessed
AFTER all cycles are complete = End point PCR
Reaction products are frequently assessed using gel
electrophoresis
End point PCR is useful for qualitative analyses, such as
determining presence/absence of target DNA in a sample
(e.g. presence/absence of HIV provirus in blood sample)
End point PCR is NOT the method of choice for
quantitative analyses, where want to determine the quantity
of target DNA in samples (e.g. HIV levels in a blood sample)
- PCR reactions are usually not in the exponential phase of
amplification at the end of all cycles
- Therefore, the level of amplified product is not directly related to the
level of target in the starting sample
Patient #1 Patient #2 Patient #3
HIV- HIV+ HIV+
0.1% of cells infected 10% of cells infected
Draw Blood WBCs WBCs WBCs
Isolate DNA
ASSESS PCR
PRODUCTS
End Point PCR AT THE END OF
AMPLIFICATION
Gel Electrophoresis
PCR
products
bands
not proportional
to amount of target
QPCR
Quantitative real time PCR (QPCR) assesses PCR
products DURING the amplification process
QPCR utilizes a reporter molecule whose level of
fluorescence is directly proportional to the amount of
amplified DNA in a sample
Capturing data throughout the PCR reaction allows for the
measurement of fluorescence during the exponential phase
of amplification, when the level of amplified product is
directly proportional to the level of target DNA in the starting
sample
Reporter Systems for QPCR
A. Probe-based reporter system (TaqMan)
A Reporter
B
probe Report
R dye
PCR primer
R
R Q R R
5 5 3 R
3 5 DNA target 3
Polymerase
R
R R
R
PCR primer
Q
5 3 5
3 5 DNA target 3 R R
Reporter (R) fluoresces when released from Reporter (R) fluoresces when
quencher (Q) by 5->3 exonuclease activity bound to double-stranded DNA
of polymerase
Reporter Systems for QPCR
B. Dye-based reporter system
Reporter
B
probe Reporter
R dye
R
R Q R R
5 3 R
5 DNA target 3 5 DNA target
se
R
R R
R
PCR primer
Q
3 5 3
5 DNA target 3 R R 5 DNA target
resces when released from Reporter (R) fluoresces when
5->3 exonuclease activity bound to double-stranded DNA
polymerase
Threshold Cycle (CT or CQ)
In QPCR, a threshold level is set above the background
fluorescence, but within the exponential phase of
amplification
The threshold cycle (CT or CQ) = cycle at which
fluorescence for a given sample reaches the threshold
The higher the starting number of copies of target
DNA in a sample, the fewer cycles it takes to reach the
threshold
Comparing amplification plots for two samples will show
which sample has a higher concentration of DNA target
Amplification Plot
Fluorescence of
amplified DNA (Fluorescence vs # Cycles)
Starting number of target DNA
copies before amplification
Amplification plots for four samples:
A Amplification B Standard Curve
1) Sample with 108 target starting DNA copies
2) Sample with 107 target starting DNA copies
Relative Fluorescence Units
3000
24
Ct (Threshold Cycle)
2500 108 3) Sample with 106 target starting DNA copies
22
2000 107 Sample with 105 target starting DNA copies
4) Interpolation
20
1500
106 of unknown
1000 18
105
500
Threshold
16
CT = cycle where reaction reaches
0 threshold
14
0 5 10 15 20 25 30 5.0 5.5 6.0 6.5 7.0 7.5 8.0
For sample with 10 8 target DNA
Cycles Log Starting
~ 14 cycles (DNA Copies)
copies, CT =
Quantity
Takes fewer cycles for sample with 108 target DNA
copies to reach the threshold (CT =~ 14) than the
sample with 107 target DNA copies (CT = ~ 18)
Linear Relationship Between
CT and Log Starting Target Quantity
A Amplification Plots
Amplification B Standard Curve
Derived from Amplification Plots
Relative Fluorescence Units
3000
24
Ct (Threshold Cycle)
2500 108
22
2000 107
20
1500
106
1000 18
105
500
Threshold
16
0
14
0 5 10 15 20 25 30 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Cycles Log Starting Quantity (DNA Copies)
Unknown Quantities Can Be
Interpolated from Known Standards
(Standard Curve)
A Amplification Plots
Amplification B Standard Curve
Standard Curve
Relative Fluorescence Units
3000
24
Ct (Threshold Cycle)
2500 108
22
2000 107 Interpolation
20
1500
106 of unknown
1000 18
105
500
Threshold
16
0
14
0 5 10 15 20 25 30 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Cycles Log Starting Quantity (DNA Copies)
Unknown sample By interpolation:
with CT = 20 unknown sample has
106.5 or 3.16 x 106 target
DNA copies
(before amplification)
Melt Curves
Melt curves are initiated at the
end of the amplification cycles
PCR reactions are slowly heated
to 94oC => fluorescence drops as
DNA strands denature
The temperature at which
DNA denatures is dependent
upon the DNA length &
base composition (%GC, %AT)
A single peak in the melt curve
indicates a single species of DNA
molecule was amplified
Today:
Form into groups of three students
(Must have 8 groups total)
Each group will:
- receive three samples with unknown quantities of
target DNA
- prepare dilutions of target DNA to make a standard
curve with known quantities (Procedure 2)
- setup eight PCR reactions (Procedure 3)
Split up into 8 groups:
Group # Unknowns Group Members Names
1 A, B, C
2 B, C, D
3 C, D, E
4 D, E, F
Signup on chalkboard
5 E, F, A
6 F, A, B
7 A, B, D
8 C, E, F
and record your unknowns on p 114
Procedure 2: Group prepares serial dilutions
of target DNA
Tube #1 10 L DNA Stock + 90 L nuclease-free water = 1:10 dilution
vortex to mix
Tube #2 10 L 1:10 dilution + 90 L nuclease-free water = 1:100 dilution
vortex to mix
Tube #3 10 L 1:100 dilution + 90 L nuclease-free water = 1:1000 dilution
vortex to mix
Tube #4 10 L 1:1000 dilution + 90 L nuclease-free water = 1:10000 dilution
DNA stock is 2 X 108 target DNA copies per uL
Procedure 3: Group prepares 8 PCR reactions
NO
LABEL THE TAB TEMPLATE
AT THIS END STANDARD CURVE UNKNOWNS CONTROL
TUBE: 1 2 3 4 5 6 7 8
1:10 1:100 1:1000 1:10000 Unknown Unknown Unknown H2 O
5.0 mL Template DNA dilution dilution dilution dilution #1 #2 #3
+
15 mL PCR Master Mix:
1. Each group needs:
dNTPs
- one strip of eight tubes
Taq Polymerase : - one strip of eight caps
Forward Primer
the tab with group # at REAL
end TIME PCR:
o
Reverse Primer 2.94Label
o
C 30 seconds of strip
65 C 30 seconds 1 CYCLE Assesses Amplification
Buffer NEAREST
o TUBE
55 C 30 seconds 1 (label with black sharpie)
After EACH Cycle
MgCl2 x 30 CYCLES
SYBR Green Dye
END POINT PCR:
3. Add 15 uL master mix to each tube
Assesses Amplification
After ALL Cycles
4. Add 5 uL of each template DNA to appropriate tube
PCR Amplification & Results
When all groups have set up their reactions, your
instructor will:
- put samples in the thermocycler & run the amplification program
- instructor will e-mail 3 data files (within 48 hours):
1. .xls file with Cq values
2. .ppt file with amplification plots
3. .pdf file with melt curves
- students should use results to prepare lab report due next week
(see p. 111 for what to include in lab report)
Example of Machine Output
Sample Guide
Group Group Group Group Group Group Group Group
#1 #2 #3 #4 #5 #6 #7 #8 Instructor
1:10
1:100
Standard
Curves
1:1000
1:10000
Unknowns
No Template
Controls
.xls file- Cq results (example)
Threshold Starting
cycle quantity
.ppt file- amplification plots (example)
Group 1 Data
.ppt file- amplification plots (example)
Group 1 Data
Which plot
belongs to
which sample?
.pdf file- melt curves (example)
