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If you haven’t been impressed yet, NOW is the time! DNA Sequencing
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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 Sanger method synthesize complementary DNA strand in vitro in each tube:
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“normal” N-bases dideoxy N-bases ddA, ddC, ddG, ddT DNA polymerase primer 2 buffers & salt
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3 4
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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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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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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
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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 AP Biology Celera Genomics
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Automated Sequencing machines Really BIG labs!
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Human Genome Project U.S government project
begun in 1990
DOE & NIH
estimated to be a 15 year project 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.
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.
3. Assemble DNA sequence using overlapping sequences.
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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
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3 billion base pairs
TACGCACATTTACGTACGCGGATGCCGCGACTATGATC ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT human genome CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA 3.2 billion bases CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA TCATCCGATCAGATCATGCTAGTACATCGATCGATACT AP Biology
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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
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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
gene
RNA polypeptide 1
gene
polypeptide 2
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polypeptide 3
–
Elizabeth Pennisi, Science 2003
It’s hard to hunt for wabbits, if you don’t know what a wabbit looks like.
And we didn’t stop there…
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The Progress 122+ bacterial genomes first metazoan complete (flatworm) first eukaryote complete (yeast)
17 eukaryotic genomes complete or near completion including Homo sapiens, mouse and fruit fly
First 2 bacterial genomes complete
# of DNA base pairs (billions) in GenBank Data NCBI and TIGR APfrom Biology (www.ncbi.nlm.nih.gov and www.tigr.org )
Official “15 year” Human Genome Project: 1990-2003.
How does the human genome stack up?
Organism
Genome Size (bases) 3 billion
30,000
2.6 billion
30,000
100 million
25,000
97 million
19,000
137 million
13,000
12.1 million
6,000
4.6 million
3,200
9700
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Human (Homo sapiens) Laboratory mouse (M. musculus) Mustard weed (A. thaliana) Roundworm (C. elegans) Fruit fly (D. melanogaster) Yeast (S. cerevisiae) Bacterium (E. coli)
Estimated Genes
Human Immunodeficiency Virus (HIV) AP Biology
What have we found? When you go looking…
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…you will certainly find something!
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