MolCellEng_Notes7

MolCellEng_Notes7 - 1 Genome Sequencing 1 Background 1...

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1. Genome Sequencing 1. Background 1. Strategies and procedures for sequencing entire genomes 2. Size of human genome: 3 billion base pairs. 3. Approaches to sequence human genome involves scaling up existing techniques, developing new sequencing techniques, and using smaller genomes to start with as warm-up projects 2. Human Genome Project 1. Goals 1. Sequence entire genome, not just transcribed genes [[introns are removed and never get transcribed into mRNA – but introns still part of DNA genome!]] 2. Sequencing should be performed w/ high accuracy; 1 error in 10,000 bases 3. Develop genomic resources that would be useful for all genes, ex: collections of physical markers 4. Develop economies of scale 2. History 1. 1984 – 1 st proposoal by Hiroshima meeting 2. Human genome prj initiative by Department of Energy 1987 3. 1988 congress approval 4. 1991 initiation of project - $3 billion budget, 15 years (1991-2006); 20 institutions participated from 6 countries 1
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5. Major goals – develop proper experimental and computational tools; sequence entire genome; find all genes; store genome and gene info in public databases; discuss ethical/legal/social issues 3. Development of technology 1. 70s – Sanger sequencing, RFLP mapping, Gene cloning 2. 80s – YAC/BAC, PCR, prototype automated DNA sequencer 3. 90s – 4-color sequencing, EST sequencing, STS (Sequence tagged site) based mapping, DNA microarray 4. Scale-up of existing technologies 1. Amount of sequencing increased dramatically 2. 2000: 500-1000 kb per year (see notes for previous years) 3. All large scale sequencing is based on sanger chain termination technology 5. New technologies 1. Shot gun sequencing applied to large genomes – DNA cut into tiny pieces (blown up) and then overlapping seq. determines order 3. Sequencing Strategies 1. Automated sequencers 1. Most use sanger sequencing method 2. Fluorescently labeled reaction products 3. Capillary electrophoresis for separation 4. Most common automated sequencers – ABI & MegaBACE 2
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5. ABI 3700 1. Made by applied biosystems 2. 96 capillaries and robot loading from 384-well plates 3. Takes 2-3 hrs. per run 4. Read 600-700 bases per run 6. MegaBACE 1. Made by Amersham 2. 96 capillaries, robotic loading from 384-well plate 3. Takes 2-4 hrs. per run 4. Read 800 bases per run 4. Steps in genomic sequencing 1. Library making 1. Large-insert library from genome 2. Library of genomic fragments made in vector (BAC,PAC, or YAC) 3. Usually have several-fold coverage – every DNA seq. on five to eight diff clonse
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