A Lot More Advanced

Biotechnology Tools
Sequencing

AP Biology 2007-2008
 DNA Sequencing
 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

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 DNA Sequencing
1
 Sanger method
 synthesize
complementary DNA
strand in vitro
 in each tube:
2
 “normal” N-bases
 dideoxy N-bases
 ddA, ddC, ddG, ddT 3
 DNA polymerase
4
 primer
2
 buffers & salt

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 Reading the sequence
 Load gel with sequences from
ddA, ddT, ddC, ddG in separate
lanes
 read lanes manually & carefully
 polyacrylamide gel

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 1978 | 1980
Fred Sanger
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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 Advancements to sequencing
 Fluorescent tagging sequence data
 Computer read & analyzed

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 Advancements to sequencing
 Capillary tube electrophoresis
 no more pouring gels
 higher capacity & faster

Applied Biosystems, Inc

(ABI) built an industry on
these machines

AP Biology 384 lanes

  Big labs!
 economy of scale

PUBLIC
 Joint Genome Institute
(DOE)
 MIT
 Washington University
of St. Louis
 Baylor College of
Medicine
 Sanger Center (UK)
PRIVATE
 Celera
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Genomics
 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 entire
human 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
gov’t method
“map-based method”
1. Cut DNA segment into fragments,
arrange based on overlapping
nucleotide sequences, and clone
fragments.
2. Cut and clone into smaller fragments.
3. Assemble DNA sequence
using overlapping sequences.
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Craig Venter’s method
“shotgun method”
1. Cut DNA entire chromosome
into small fragments and clone.
2. Sequence each segment &
arrange based on overlapping
nucleotide sequences.
 Human Genome Project
On June 26, 2001, HGP published the “working
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

3G of data

AP Biology 3 billion base pairs

TACGCACATTTACGTACGCGGATGCCGCGAC
TATGATCACATAGACATGCTGTCAGCTCTAG
human genome
TAGACTAGCTGACTCGACTAGCATGATCGAT
3.2 billion bases
CAGCTACATGCTAGCACACYCGTACATCGAT
CCTGACATCGACCTGCTCGTACATGCTACTA
GCTACTGACTCATGATCCAGATCACTGAAAC
CCTAGATCGGGTACCTATTACAGTACGATCA
TCCGATCAGATCATGCTAGTACATCGATCGA
TACTGCTACTGATCTAGCTCAATCAAACTCTT
TTTGCATCATGATACTAGACTAGCTGACTGA
TCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGC
TAGTACATCGATCGATACTGCTACTGATCTA
GCTCAATCAAACTCTTTTTGCATCATGATACT
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 Raw genome data

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 NCBI GenBank
Database of
genetic
sequences
gathered
from
research

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 a
TIFF (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.”
gene protein – Elizabeth Pennisi, Science 2003

gene RNA It’s hard to

hunt for wabbits,
polypeptide 1 if you don’t know
what a wabbit
looks like.
gene polypeptide 2

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 And we didn’t stop there…

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 The Progress
4.E+10

3.E+10 122+ bacterial

genomes
3.E+10

first metazoan complete

2.E+10 (flatworm)

2.E+10 first eukaryote complete 17

(yeast) eukaryotic
1.E+10 genomes
complete or
First 2 bacterial genomes near
5.E+09 complete completion
including
0.E+00 Homo
sapiens,
Dec-82

Dec-83

Dec-84

Dec-85

mouse and
Dec-86

Dec-87

Dec-88

Dec-89

Dec-90

fruit fly
Dec-91

Dec-92

Dec-93

Dec-94

Dec-95 S1
Dec-96

Dec-97

Dec-98

Dec-99

Dec-00

Dec-01

Dec-02
# of DNA base pairs

Jun-03
(billions)
in GenBank Official “15 year”
Human Genome Project:
Data
APfrom NCBI and TIGR
Biology 1990-2003.
(www.ncbi.nlm.nih.gov and www.tigr.org )
 How does the human genome stack up?

Genome Size Estimated

Organism (bases) Genes
Human (Homo sapiens) 3 billion 30,000
Laboratory mouse (M. musculus) 2.6 billion 30,000
Mustard weed (A. thaliana) 100 million 25,000
Roundworm (C. elegans) 97 million 19,000
Fruit fly (D. melanogaster) 137 million 13,000
Yeast (S. cerevisiae) 12.1 million 6,000
Bacterium (E. coli) 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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