Learning Objectives
• Draw or model the first three cycles of PCR, including the
correct directionality (5’- and 3’-ends) of the primers and
single-stranded PCR products.
• Diagram how single-stranded products from the first cycle
of PCR are used as templates for subsequent PCR cycles.
• Demonstrate which parts of the primers will anneal to the
original DNA template and subsequent PCR products.
• Model and demonstrate when the primer restriction enzyme
sites are incorporated into double-stranded PCR products.
• Calculate the number of desired-length PCR products and
long PCR products for each amplification cycle.
• Demonstrate how the incorporation of primer restriction
enzyme sites into PCR products is a useful tool for
subsequent cloning of the product into a vector.
1
PCR = in vitro
synthesis of DNA
by thermostable
DNA polymerase
Uses?
Amplification:
exponential or
linear?
http://schoolworkhelper.net/pcr-uses-steps-purpose/
Amplify ~1 million-fold after 20 cycles
2
One cycle: 3 steps
(times may vary)
• Denaturation: 95oC, 30 sec
• Annealing: variable temp: 45-65oC, 30 sec
• Extension: 68-72oC, time: 60 sec
3
http://biosistemika.com/workshops/qpcr-basics/
Compare PCR to DNA replication in a cell
Enzyme used?
DNA Replication in the cell
PCR in a test tube
DNA polymerase
Heat resistant DNA
polymerase
Template needed?
What is composition
of template?
Primer needed? What
is composition of
primer?
Synthesis direction?
Nucleotides needed?
Which ones?
What separates
template strands?
Is entire template
replicated?
4
In-class Activity
Scenario:
Each group has been assigned a gene
to amplify from the chromosome of
Whodatamonas saintsatium, a newly
discovered (obviously fictitious)
bacterium that helps grown men win
football games on Sunday.
We have isolated “chromosomal DNA”
for you because we are such wonderful
instructors.
5
Objective: Demonstrate the 3 steps
of a PCR reaction for 3 cycles
• PCR components (in “toolkit” envelope)
• Double-stranded chromosomal DNA = twisted yarn
– Black stripes show fragment to be amplified
• Primers = 3-inch colored Bendaroos (flexible,
waxed sticks)
– Forward and reverse primers – different colors
– Restriction enzyme site to be added: yellow
piece at end of colored Bendaroo
• Newly synthesized DNA = 3 sheets of bendaroos –
different colors for different cycles
• Polymerase = your hands
• (What components are we missing?)
6
Homework
Cycle Number of
new singlestranded
products
generated
during
each cycle
1
2
Total number
of singlestranded
products
(cumulative
cycles)
Total number of
single-stranded
products of
correct length that
include the
restriction enzyme
site
2
0
Total
number of
singlestranded
products
that are too
long
2
Percentage of
correct length
products/total
products
0%
2
3
4
5
6
7
Homework Answer Key
Cycle Number of
new singlestranded
products
generated
during
each cycle
Total number
of singlestranded
products
(cumulative
cycles)
Total number of
single-stranded
products of
correct length that
include the
restriction enzyme
site
Total
number of
singlestranded
products
that are too
long
Percentage of
correct length
products/total
products
1
2
2
0
2
0%
2
4
6
2
4
33%
3
8
14
8
6
57%
4
16
30
22
8
73%
5
32
62
52
10
84%
6
64
126
114
12
90%
8