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)