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8/8/2019 Biotechnology-4 (KFolger)
http://slidepdf.com/reader/full/biotechnology-4-kfolger 1/25
AP Biology 2007-2008
A Lot More Advanced
Biotechnology Tools
Sequencing
8/8/2019 Biotechnology-4 (KFolger)
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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
8/8/2019 Biotechnology-4 (KFolger)
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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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AP Biology
Reading the sequence
Load gel with sequences fromddA, ddT, ddC, ddG in separatelanes
read lanes manually & carefully
polyacrylamide gel
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AP Biology
Fred Sanger 1978 | 1980
This was his 2nd Nobel Prize!!
1st was in 1958 for the
structure of insulin
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AP Biology
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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AP Biology
Advancements to sequencing
Fluorescent tagging sequence data
Computer read & analyzed
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AP Biology
Applied Biosystems, Inc
(ABI) built an industry on
these machines
Advancements to sequencing
Capillary tube electrophoresis
no more pouring gels
higher capacity & faster
384 lanes
8/8/2019 Biotechnology-4 (KFolger)
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AP Biology
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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AP Biology
Automated Sequencing machines
Really BIG labs!
8/8/2019 Biotechnology-4 (KFolger)
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AP Biology
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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AP Biology
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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AP Biology
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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AP Biology
Sequence of
46 Human Chromosomes
3 billion base pairs
3G of data
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AP Biology
TACGCACATTTACGTACGCGGATGCCGCGACTATGATC
ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT
CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC
GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA
CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA
GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT
CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC
TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC
TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC
TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG
ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA
TCATCCGATCAGATCATGCTAGTACATCGATCGATACT
human genome3.2 billion bases
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AP Biology
Raw genome data
8/8/2019 Biotechnology-4 (KFolger)
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AP Biology
NCBI GenBank
Database of
genetic
sequences
gathered
fromresearch
Publicly
available on
Web!
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AP Biology
Organizing the data
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AP Biology
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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AP Biology
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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AP Biology
And we didn¶t stop there«
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AP Biology
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
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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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AP Biology
What have we found?
When you go looking«