A Lot More Advanced Biotechnology Tools (Part 1)
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2007-2008
A Lot More Advanced Biotechnology Tools(Part 1)
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
2
1
3
42
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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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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.