Invented by Kary Mullis
Mullis and Faloona, 1987. Specificsynthesis of DNA in vitro via apolymerase-catalyzed chain reaction.
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
Specifically targets and amplifies a SINGLE sequence from within a complex mixture of DNA.
How is this different from cloning?
Takes advantage of basic requirements of replication
A DNA template
Nucleotides
Primers
polymerase
PCR is DNA replication in a test tube
Primers
Must have some information about sequence flanking your target
Primers provide specificity
Complementary to opposite strands with 3’ends pointing towards each other
Should have similar melting temperatures
Be in vast excess
Melting temperature
Tm ℃= 2(A/T) 4(G/C)
Tm ℃ Temperature at which half possible H bonds are formed
The basic process
Thermocycling
94 degrees
55 degrees
70 degree
Heat-stable polymerase is vital to the ease of the process…
嗜热水生菌(Thermus aquaticus)
-The Thermus aquaticus DNA polymerase
-Taq
-Not permanently destroyed at 94ºC
-Optimal temperature is 72ºC
Problems with Taq
-Does not have proof readng ability
-Error rate 1 in 2 X 104 bases
-Seems rare but can be recovered in cloning a single molecule
-Newer polymerases have high fidelity
Termplates for PCR
-Small amount of template
-In theory a single molecule
-Do not need to isolate sequence of interest
-DNA template need not be highly purified
-DNA is stable in absence of nucleases
-Dried blood
-Semen stains
Dried blood
Semen stains
Vaginal swabs
Single hair
Fingernail scrapings
Insects in Amber
Egyptian mummies
Buccal Swab
Toothbrushes
PCR variations
Cloning PCR Fragments
Taq leaves 3’ A overhang.
rtPCR
-Reverse trancriptase
-Use mRNA as a template
-Isolated cDNA clones
-Can be quantitative
Inverse PCR
Nested primers
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