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Bio/Chem 475 Then, PCR Optimization. Polymerase Chain Reaction , Voet &Voet, Ch. 5, pp. 113- PCR Primer Design, Haggard 112, this week, PCR next week, Kary B. Mullis Nobel Laureate, 1993 Chemistry P olymerase C hain R eaction

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Presentation Transcript
slide2

Then,

  • PCR Optimization.
  • Polymerase Chain Reaction,
      • Voet &Voet, Ch. 5, pp. 113-
  • PCR Primer Design,
      • Haggard 112, this week,
  • PCR next week,
p olymerase c hain r eaction

Kary B. Mullis

Nobel Laureate, 1993

Chemistry

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!"

slide4
"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
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

oligonucleotides specific primers
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 primer

Making One Strand Of DNA

Add Polymerase

Add dNTPs

making two more strands
Making Two More Strands

Must Denature

Separate Strands

denaturing can t use helicase in vitro
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 strands11

5’--GCATGCATTAG

GCTACATCGACATCGACTAGCACTG--3’

add primer to second strand

Making Two More Strands

3’--GCTACGTAATCCGATGTAGCTGTAG

CTGATCGTGAC--5’

5’--GCATGCATTAG

GCTACATCGACATCGACTAGCACTG--3’

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

Add polymerase, ect.

denaturation step bad
Denaturation Step Bad

…several rounds of in vitro replication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions.

slide13

…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
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
Click

…Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase,

… Thermus aquaticus: Taq polymerase.

slide16

5’--GCATGCATTAT

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

CTGATCGTGAC--5’

CTGATCGTGAC--5’

5’--GCATGCATTAT

Denature Step

~30 seconds

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

Annealing Step

~30 seconds

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

CTGATCGTGAC--5’

5’--GCATGCATTAT

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

94o

Synthesis

~30 seconds/kb

72o

PCR

~60o

exponential synthesis
Exponential Synthesis
  • as few as 1 DNA templates required,
  • excess dNTPS,
  • excess primers,
  • multiple cycles.