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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
synthesize complementary DNA strand in vitro
in each tube: “normal” N-bases dideoxy N-bases
ddA, ddC, ddG, ddT
DNA polymerase primer buffers & salt
2
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AP Biology
Reading the sequence Load gel with sequences from
ddA, ddT, ddC, ddG in separate lanes read lanes manually & carefully polyacrylamide gel
AP Biology
Fred Sanger1978 | 1980
This was his 2nd Nobel Prize!! 1st was in 1958 for the
structure of insulin
AP Biology
Advancements to sequencing Fluorescent tagging
no more radioactivity all 4 bases in 1 lane
each base a different color
Automated reading
AP Biology
Advancements to sequencing Fluorescent tagging sequence data Computer read & analyzed
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
AP Biology
PUBLIC Joint Genome Institute
(DOE) MIT Washington University
of St. Louis Baylor College of
Medicine Sanger Center (UK)PRIVATE Celera Genomics
Big labs! economy of scale
AP Biology
Automated Sequencing machines Really BIG labs!
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 entire human genome 3 billion base pairs
Celera Genomics Craig Venter challenged gov’t would do it faster, cheaper private company
AP Biology
Different approaches
3. Assemble DNA sequence using overlapping sequences.
“map-based method”gov’t method
“shotgun method”Craig Venter’s method
1. Cut DNA entire chromosome into small fragments and clone.
2. Sequence each segment & arrange based on overlapping nucleotide sequences.
1. Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments.
2. Cut and clone into smaller fragments.
AP Biology
Human Genome ProjectOn 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
AP Biology
Sequence of 46 Human Chromosomes
3 billion base pairs
3G of data
AP Biology
TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT
human genome3.2 billion
bases
AP Biology
Raw genome data
AP Biology
NCBI GenBankDatabase of genetic sequences gathered from research
Publicly available on Web!
AP Biology
Organizing the data
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.
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.”
– Elizabeth Pennisi, Science 2003
gene
polypeptide 1
polypeptide 2
polypeptide 3
proteingene
It’s hard tohunt for wabbits,if you don’t know
what a wabbitlooks like.
RNAgene
AP Biology
And we didn’t stop there…
AP Biology
The ProgressD
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5.E+09
1.E+10
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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 Homo sapiens, mouse and fruit fly
Official “15 year”Human Genome Project:
1990-2003.
# of DNA base pairs (billions)
in GenBank
AP Biology
How does the human genome stack up?
OrganismGenome Size
(bases)Estimated
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 3,200Human Immunodeficiency Virus (HIV) 9700 9
AP Biology
What have we found? When you go looking…
AP Biology
…you will certainly find something!