Ex. 26: DNA Amplification of
Mycobacterium tuberculosis by
PCR is DNA replication in a test tube
Objectives ??
Invented by Kary Mullis, 1983.
Nobel Prize 1993.
“I was working for Cetus, making
oligonucleotides. They were heady
times. Biotechnology was in flower and
one spring night while the California
buckeyes were also in flower I came
across the polymerase chain reaction. I
was driving with Jennifer Barnett to a
cabin I had been building in northern
California. She and I had worked and
lived together for two years. She was
an inspiration to me during that time as
only a woman with brains, in the bloom
of her womanhood, can be. That
morning she had no idea what had just
happened. I had an inkling. It was the
first day of the rest of my life.”
- from Karry Mullis’s autobiography at
the Nobel e-Museum
Did He Really Invent PCR?
The basic principle of replicating a piece of
DNA using two primers had already been
described by Gobind Khorana in 1971:
– Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.
Progress was limited by primer synthesis
and polymerase purification issues.
Mullis properly exploited amplification.
What is the Polymerase Chain
Reaction?
• Think of it as a molecular photocopier
• It is a means of selectively amplifying a
particular segment of DNA.
• The segment may represent a small
part of a large and complex mixture of
DNAs: e.g. a specific exon of a human
gene.
A Molecular Photocopier
A photocopier capable of duplicating a part of a
sentence:
“The next day was quite a different day.
Instead of being hot and sunny, it was cool
and misty. Pooh didn’t mind for himself, but
when he thought of all the honey the bees
wouldn’t be making, a cold misty day always
made him feel sorry for them.” A.A. Milne,
1928.
The words in blue must be unique for the copier to
locate the correct piece of text.
How Powerful is PCR?
PCR can amplify a single DNA molecule, e.g.
from a single sperm.
PCR can amplify DNA to a usable amount
(visible by gel electrophoresis) in ~2 hours.
The template DNA need not be highly purified
— a boiled bacterial colony.
The PCR product can be digested with
restriction enzymes, sequenced or cloned.
The Basics of PCR Cycling
30 - 35 cycles, each comprising:
– Denaturation (95°C), 30 sec.
– Annealing (55–60°C), 30 sec.
– Extension (72°C),
time depends on
product size.
DolanDNA
Learning Center:
PCR Tutorial
DolanDNA
Learning Center
on YouTube
Additional
narrated PCR
Tutorial
What’s in the Reaction?
1. Template DNA
2. Reaction buffer and magnesium
ions
3. Nucleotides (dNTPs)
4. Primers
5. DNA polymerase (usually Taq)
So Then, it’s Easy?
Cycling performed with three water baths.
Thermal cyclers introduced in 1986.
Early polymerases were not thermostable, so
had to be replenished each cycle.
The 37°C temperature caused non-specific
priming, resulting in unwanted products.
Taq (Thermus aquaticus) DNA
polymerase first described in 1988.
Forensic Microbiology
• Primer for a specific organism will
allow for detection that particular
organism
RT-PCR with a norovirus
primer
• Real-time PCR: Newly made
DNA tagged with a fluorescent
dye; the levels of fluorescence
can be measured after every
PCR cycle
• Reverse-transcription PCR
(RT-PCR): Reverse transcriptase
makes DNA from viral RNA or
mRNA
Textbook Clinical Focus, p. 266
Day 1
Materials needed per 2 tables:
Four 200 µl PCR tubes containing PCR beads
Micropipette and tips capable of measuring out 10 µl
Patient samples
Positive control sample
Distilled water for negative control
Equipment: Nano centrifuge, PCR Thermocycler
Work in a team of 8 students.
Part 2 of Day 1
Cast, Load, and Electrophorese a 1.5 %
Agarose Gel
Materials needed per table:
 Amplified PCR products from
last session
 Bottle of 0.5 X TBE (Trisborate/EDTA) buffer
 50 ml measuring cylinder,
250ml Erlenmeyer flask
 Minigel electrophoresis
apparatus with gel tray, spacers,
and comb
 micropipettes and matching tips
 Microcentrifuge tube containing
20 µl of 10x gel loading buffer.
Materials shared by whole class:
1. Scales, flasks, electrophoresis
grade agarose, cylinder, and TBE
buffer at “gel making station”
2. Microwave
3. Power source (4 available per
class)
4. Nanofuge (2 per room)
5. DNA standard (instructor handles
it)
6. “InstaStain” ethidium bromide
staining sheets (handled or
supervised by the instructor).
7. Gel documentation system
Understanding Gel
Electrophoresis
Make 30 ml of a 1.5% agarose gel.
How?
How does gel electrophoresis separate
DNA fragments?
• Gel acts as sieve to filter DNA fragments
• DNA fragments are naturally negatively charged
(phosphate backbone)
• DNA pulled towards anode (pos. electrode) by
electric attraction
• Smaller fragments move faster through the gel
and larger fragments move slower
• gel electrophoresis is optimized by adjusting
agarose concentration in gel
+ + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + +
DNA is negatively
charged and
therefore repelled
from the negative
pole and attracted
towards the
positive pole
--------------- ------ --- --- --- ---- -
+ + + + + + + + + + + + +
_ _ _ _ _ _ _ _ _ _ _ _
A Typical Image of an Agarose Gel
Under UV Light
Decreasing
DNA
Size
Largest DNA fragments
Smallest DNA fragments
The Intensity of the Band is Proportional
to the Concentration Of DNA
• An important point to remember is that the
intensity of the band is proportional to the
amount of DNA found in the band
The upper band has far less
DNA when compared to the lower
band. The intensity of the bands
are proportional to the amount of
DNA at that position in the gel
Sizing The Fragments of DNA
The sizes of the various fragments can be
identified by including a “ladder” in the gel
– A ladder is a mixture of DNA fragments of known
size
– A ladder is usually run beside the unknown
sample so that the sizes
of the various DNA
fragments in the sample
can be identified
Marker / Ladder / Size Standard
• Mixture of DNA fragments of
selected sizes
• When run in a gel, fragments will
separate into distinct bands that
can be used as references
• Fragment size always stated as [X]
base pairs (bp)
• Two common ladders: 200bp and
1kbp (1000 bp) ladders
• 200 bp ladder composed of a
mixture of small fragments (200 to
4000 bp)
• Ladders commercially available
Sizing a Gel Product
Base
Pairs
(bp)
4000
3000
2000
1600
2000 bp
1000 bp
1000
4000
3000
500
2000
1600
1Kbp
Sample
1000
ladder
500
Size of this Fragment?
Sample 1Kbp
ladder
The size of the fragment is…??
Size In Base Pair
(bp)
1500
1200
1000
600
500
400
300
200
100
1 kbp ladder was used
?
4000
3000
2000
1600
1000
500
Music video from "Scientists for Better PCR" and Bio-Rad.