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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

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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.