Operator Promoter
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Transcript of Operator Promoter
Operator Promoter
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Nobel Prize – 1958 – Developed method to sequence proteins (insulin)
Nobel Prize – 1980 – Developed method to sequence DNA (bacteriophage, human mitochondria)
Dr. Fred Sanger
OH
Deoxynucleoside triphosphate
(ddNTP)
Dideoxynucleoside triphosphate
(ddNTP)
Radioactivity
X-ray
Operator Promoter
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T7 Primer = TAATACGACTCACTATAGGGSP6 Primer = GATTTAGGTGACACTATAG
Sequence from an unknown piece of DNA. A single sequencing reaction was loaded into four lanes, allowed to run for a couple hours (right set of 4 lanes; called the long run) and then more of the same reaction was loaded and run for another two hours (left set of 4 lanes; called the short run).
This means that someone has to “read” all of these gels
Human Genome ProjectFirst discussed at a Dept. of Energy meeting (1984) on the effects
of radiation on U.S. populations
1988 = Congress authorized Dept. of Energy & Nat. Institute of Healthto use $3 billion over a 15-year period
1990 = it started…predicted to be finished in 2005At a cost of $3 billion (about $1 per base)
Dr. Francis CollinsNat. Institute of Health
Dr. J. Craig VenterCEO, Celera Genomics Corp.
……
“Rough map of human genome completed”Milestone in genetics expected to be giant boon for medicine
June 26, 2000
How did it get done 5 years early?
Capillary Gel Electrophoresis…allowed people to bypass
having to do slab-gel electrophoresis
…and now there is “Multichannel” so can do
8 at once
Rooms full of sequencers
…and robotics….
Primer Walking:
GCGCGATATATCGCGTGCGTAAAGGCTCGA
TCGATTCGAGTTATT TATTCCACGTAGCAGC
GCGCGATATATCGCGTAAAGGCTCGATTCGAGTTATTCCACGTAGCAGC
Dr. J. Craig Venter originally worked for the governmenton the project, but disagreed how it should work.
He wanted to use the “shotgun method”…chopping the entire genome into small pieces, sequencing them, and then fitting them together like pieces of a puzzle. He also integrated Capillary Gel Electrophoresis and the use of robotics and computers. He quit, and got the financial backing to found Celera Genomics Corp.
The idea was to “scoop” the feds and sequence the genome on their own.
This raised lots of questions about the marketability of the humangenome sequence.
GGGTACACATAGCT
TAGCTCTCGAGAGG
GAGGCTCTCTCTCT
TTTTTGGGTA
GGGTACACATAGCTTAGCTCTCGAGAGG
GAGGCTCTCTCTCT
TTTTTGGGTA
This method uses Genomic Libraries made with different restriction enzymes. They just used regular
sequencing primers (like T7 and SP6).
Shotgun Method is heavily dependent on computers!
TTTTTGGGTACACATAGCTCTCGAGAGGCTCTCTCTCT
Ventner also sequenced human cDNAs (mRNA).
This meant that they could really focus on the parts of the Genome that actually encode gene product rather methodically Primer-walking through the genome!
The part of the genome that took longer to complete were the parts with lots of repetitive sequences:
TTAGGGTTAGGG
TTTTTTATTTTTT
TTAGGCAGCAGCAGCATTTTCTTTT
TTAGGGTTAGGG
It was totally completed in 2003
Genomes that are 100% completed:
Prokaryotes = 302 Fungi = 9Protists = 3Plants = 2 (Arabidopsis, rice)Animals = 4 (fruitfly, nematode, mouse, human)
• 272 more genomes published as “drafts”• 505 other genomes “in progress”
= 1097 genome projects in all
Celera’s stock dropped the day of the joint announcement about finishing the genome project.
Celera is hoping to make money by leasing genome analysis software and on patents on genes of other species like bacteria, fruitfly, dogs, etc.