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DNA Sequencing

DNA Sequencing. DNA sequencing. How we obtain the sequence of nucleotides of a species. …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…. Which representative of the species?. Which human?

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DNA Sequencing

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  1. DNA Sequencing

  2. DNA sequencing How we obtain the sequence of nucleotides of a species …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

  3. Which representative of the species? Which human? Answer one: Answer two: it doesn’t matter Polymorphism rate: number of letter changes between two different members of a species Humans: ~1/1,000 Other organisms have much higher polymorphism rates • Population size!

  4. Why humans are so similar Out of Africa N A small population that interbred reduced the genetic variation Out of Africa ~ 40,000 years ago Heterozygosity: H H = 4Nu/(1 + 4Nu) u ~ 10-8, N ~ 104  H ~ 410-4

  5. Human population migrations • Out of Africa, Replacement • “Grandma” of all humans (Eve) ~150,000yr • Ancestor of all mtDNA • “Grandpa” of all humans (Adam) ~100,000yr • Ancestor of all Y-chromosomes • Multiregional Evolution • Fossil records show a continuous change of morphological features • Proponents of the theory doubt mtDNA and other genetic evidence • New fossil records support out of Africa • Good popular article in Economist on that http://www.economist.com/science/displaystory.cfm?story_id=9507453

  6. DNA Sequencing – Overview 1975 • Gel electrophoresis • Old technology by F. Sanger • Newer sequencing technologies • Whole genome strategies • Physical mapping • Shotgun sequencing • Computational fragment assembly • Future variants of sequencing • Resequencing of humans • Microbial and environmental sequencing • Cancer genome sequencing 2015

  7. DNA Sequencing Goal: Find the complete sequence of A, C, G, T’s in DNA Challenge: There is no machine that takes long DNA as an input, and gives the complete sequence as output Can only sequence ~900 letters at a time

  8. DNA Sequencing – vectors DNA Shake DNA fragments Known location (restriction site) Vector Circular genome (bacterium, plasmid) + =

  9. Different types of vectors

  10. DNA Sequencing – gel electrophoresis • Start at primer (restriction site) • Grow DNA chain • Include dideoxynucleoside (modified a, c, g, t) • Stops reaction at all possible points • Separate products with length, using gel electrophoresis

  11. Pyrosequencing / 454 Image credits: 454 Life Sciences

  12. Solexa / ABI SOLiD Image credits: Illumina, Applied Biosystems

  13. Illumina / Affymetrix genotyping Image credits: Illumina, Affymetrix

  14. Growth of DNA Sequencing Capacity Human Genome Project (1990 – 2003) $3 billion 3 Gbp 2008 100,000 Gbp 2010 500,000 Gbp 2011 5,000,000 Gbp

  15. Sequencing Applications 100 million species Each individualhas different DNA Within individual, some cells have different DNA(i.e. cancer)

  16. Sequencing Applications What genes are on/off, when, and in which cells? Where do moleculesbind to DNA?

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