Transcript Making DNA: Components - Western Washington University

Bio/Chem 475
• Polymerase Chain Reaction,
• Voet &Voet, Ch. 5, pp. 113-
• PCR Primer Design,
• Haggard 112, this week,
• PCR next week,
Then,
• PCR Optimization.
Polymerase Chain Reaction
…invented by Kary Mullis
while cruising in a Honda
Civic on Highway 128
from San Francisco to
Mendocino,
"It was quiet and something
just went, Click!"
Kary B. Mullis
Nobel Laureate, 1993
Chemistry
"THE SUN HAD been hot that day in Mendocino County. A dry wind
had come out of the east, and nobody knew how hot it had been
until, around sunset, the wind stopped. I drove up from Berkeley
through Cloverdale headed to Anderson Valley. The California
buckeyes poked heavy blossoms out into Highway 128. The pink
and white stalks hanging down into my headlights looked cold, but
they were loaded with warmed oils that dominated the dimension
of smell.
It seemed to be the night of the buckeyes, but something else was
stirring.""My little silver Honda's front tires pulled us through the
mountains. My hands felt the road and the turns. My mind drifted
back to the lab. DNA chains coiled and floated. Lurid blue and
pink images of electric molecules injected themselves somewhere
between the mountain road and my eyes."
Opening words, Dancing Naked in the Mind Field, © 1998, by Dr. Kary Mullis, Pantheon Books.
Mullis…
... “PCR is a chemical procedure that will
make the structures of the molecules of
our genes as easy to see as billboards in
the desert and as easy to manipulate as
Tinkertoys”.
DNA
Making DNA: Components
Cell
PCR
ss DNA
template
dNTPs
helicase, etc.
?
present
present
Primer
primase
?
DNA
Polymerase
DNA
polIII
nucleus
?
Environment
test tube
Oligonucleotides
specific primers
...short pieces of synthetic DNA can be
manufactured that contain any sequence,
…template specific!
~ Odds of a
Specific Sequence
20-mer: 9.1 x 10-13
Making One Strand Of DNA
add primer
Add Polymerase
Add dNTPs
Making Two More Strands
Must Denature
Separate Strands
Denaturing
can’t use helicase in vitro
…DNA denaturing conditions such as high
heat or low salt concentrations irreversibly
denature or inactivate most polymerases,
…dNTPs are not affected by denaturation,
…primers are not affected by denaturation.
Making Two More Strands
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
add primer to second strand
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
Add polymerase, ect.
Denaturation Step Bad
…several rounds of in vitro replication could
be performed (prior to PCR), however,
accumulation of denatured polymerases
quickly poisoned the reactions.
…bacteria discovered in a hot spring in Yellowstone Natural Park in
1965,
…lives in salty water that ranges from 70o - 75o C,
…thus, does DNA replication at very high temperatures.
Thermus aquaticus’ Enzymes
…basic research demonstrated that many
enzymes isolated from Thermus aquaticus
function at very high temperatures,
…temperatures nearing 100o C,
…DNA denaturating temperatures.
Click
…Kary Mullis realized that repetitive
rounds of DNA synthesis could be
performed by using a heat-stable
polymerase,
… Thermus aquaticus: Taq polymerase.
5’--GCATGCATTAT
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
CTGATCGTGAC--5’
94
Synthesis
~30 seconds/kb
72o
o
Denature Step
~30 seconds
PCR
~60o
Annealing Step
~30 seconds
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
CTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
CTGATCGTGAC--5’
5’--GCATGCATTAT
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAT
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
Exponential Synthesis
• as few as 1 DNA templates required,
• excess dNTPS,
• excess primers,
• multiple cycles.
QuickTime™ and a
Cinepak decompressor
are needed to see this picture.