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AP Biology 2007-2008

A Lot More Advanced

Biotechnology Tools

Sequencing



AP Biology

Sanger method

determine the base sequence of DNA

based on replication

dideoxynucleotides

ddATP, ddGTP, ddTTP, ddCTP

missing O for bonding of next

nucleotide

terminates the growing chain

DNA Sequencing



AP Biology

DNA Sequencing

Sanger method

synthesizecomplementary DNAstrand in vitro

in each tube:

³normal´ N-bases

dideoxy N-bases

ddA, ddC, ddG, ddT

DNA polymerase

primer

buffers & salt

2

1

3

4

2



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Reading the sequence

Load gel with sequences fromddA, ddT, ddC, ddG in separatelanes

read lanes manually & carefully

polyacrylamide gel



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Fred Sanger 1978 | 1980

This was his 2nd Nobel Prize!!

1st was in 1958 for the

structure of insulin



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Advancements to sequencing

Fluorescent tagging

no more radioactivity

all 4 bases in 1 lane

each base a different color Automated reading



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Fluorescent tagging sequence data

Computer read & analyzed



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Applied Biosystems, Inc

(ABI) built an industry on

these machines


Capillary tube electrophoresis

no more pouring gels

higher capacity & faster

384 lanes



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PUBLIC

Joint Genome Institute(DOE)

MIT

Washington Universityof St. Louis

Baylor College of Medicine

Sanger Center (UK)

PRIVATE

Celera Genomics

Big labs!

economy of scale



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Automated Sequencing machines

Really BIG labs!



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Human Genome Project

U.S government project

begun in 1990 estimated to be a 15 year project

DOE & NIH

initiated by Jim Watson led by Francis Collins

goal was to sequence entirehuman genome 3 billion base pairs

Celera Genomics Craig Venter challenged gov¶t

would do it faster, cheaper

private company



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Different approaches

3. Assemble DNA sequenceusing overlapping sequences.

³map-based method´gov¶t method

³shotgun method´Craig Venter¶s method

1. Cut DNA entire chromosome

into small fragments and clone.

2. Sequence each segment &

arrange based on overlappingnucleotide sequences.

1. Cut DNA segment into fragments,

arrange based on overlapping

nucleotide sequences, and clone

fragments.2. Cut and clone into smaller fragments.



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Human Genome Project

On June 26, 2001, HGP published the ³working

draft´ of the DNA sequence of the human genome.

Historic Event!blueprint

of a human

the potential to

change science &

medicine



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Sequence of

46 Human Chromosomes

3 billion base pairs

3G of data



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TACGCACATTTACGTACGCGGATGCCGCGACTATGATC

ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT

CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC

GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA

CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA

GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT

CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC

TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC

TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT

ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA

TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC

TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG

ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA

TCATCCGATCAGATCATGCTAGTACATCGATCGATACT

human genome3.2 billion bases



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Raw genome data



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NCBI GenBank

Database of

genetic

sequences

gathered

fromresearch

Publicly

available on

Web!



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Organizing the data



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Maps of human genes«

Where the genes are«

mapping genes & their mutant alleles

QuickTime and aTIFF (Uncompressed) decompressor

are needed to see this picture.



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Defining a gene«

´Defining a gene is problematic because«

one gene can code for several protein products,

some genes code only for RNA, two genes can

overlap, and there are many other complications.µ ± El izabeth Pennisi, Science 2003

gene

polypeptide 1

polypeptide 2

polypeptide 3

proteingene

It·s hard to

hunt for wabbits,if you don·t know what a wabbit

looks like.

RNAgene



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And we didn¶t stop there«



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The Progress

D e c - 8 2

D e c - 8 3

D e c - 8 4

D e c - 8 5

D e c - 8 6

D e c - 8 7

D e c - 8 8

D e c - 8 9

D e c - 9 0

D e c - 9 1

D e c - 9 2

D e c - 9 3

D e c - 9 4

D e c - 9 5

D e c - 9 6

D e c - 9 7

D e c - 9 8

D e c - 9 9

D e c - 0 0

D e c - 0 1

D e c - 0 2

J u n - 0 3

S1

0.E+00

5.E+09

1.E+10

2.E+10

2.E+10

3.E+10

3.E+10

4.E+10

First 2 bacterial genomes

complete

122+ bacterial

genomes

Data from NCBI and TIGR(www.ncbi.nlm.nih.gov and www.tigr.org )

first eukaryote complete

(yeast)

first metazoan complete

(flatworm)

17

eukaryotic

genomes

complete or

near

completion

including

H omo

sapiens,mouse and

fruit fly

Official ³15 year´

Human Genome Project:

1990-2003.

# of DNA base pairs

(billions)

in GenBank



AP Biology

How does the human genome stack up?

Organism

Genome Size

(bases)

Estimated

Genes

Human (H omo sapiens) 3 billion 30,000Laboratory mouse (M. muscu l us) 2.6 billion 30,000

Mustard weed ( A. thal iana) 100 million 25,000

Roundworm (C. el egans) 97 million 19,000

Fruit fly (D. mel anogaster )

137 million 13,000 Yeast (S. cerevisiae) 12.1 million 6,000

Bacterium (E . col i ) 4.6 million 3,200

Human Immunodeficiency Virus

(HIV)9700 9



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What have we found?

When you go looking«



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«you will certainly find something!

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