AP Biology 2007-2008

A Lot More Advanced Biotechnology Tools

Sequencing

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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

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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

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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 Sanger1978 | 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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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

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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

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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 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

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.

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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

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Sequence of 46 Human Chromosomes

3 billion base pairs

3G of data

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TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT

human genome3.2 billion

bases

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Raw genome data

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NCBI GenBankDatabase 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 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.”

– 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

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And we didn’t stop there…

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The ProgressD

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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

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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

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What have we found? When you go looking…

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…you will certainly find something!