Bio/Chem 475

• Polymerase Chain Reaction,• Voet &Voet, Ch. 5, pp. 113-

• PCR Primer Design,• Haggard 112, this week,

• PCR next week,

• PCR Optimization.Then,

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. MullisNobel 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

helicase, etc. ?

dNTPs present present

Primer primase ?

DNA Polymerase

DNA

polIII

?

Environment nucleus test tube

Oligonucleotidesspecific 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 Polymerase

Add dNTPs

add primer

Making Two More Strands

Must DenatureSeparate Strands

Denaturingcan’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

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAG

CTGATCGTGAC--5’

GCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAG

Add polymerase, ect.

add primer to second strand

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.

94o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Denature Step~30 seconds

~60o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Annealing Step~30 seconds

72o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

Synthesis~30 seconds/kb

PCR

Exponential Synthesis

• as few as 1 DNA templates required,

• excess dNTPS,

• excess primers,

• multiple cycles.

QuickTime™ and aCinepak decompressor

are needed to see this picture.