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DNA Sequencing Methods

Emily Buckhouse. DNA Sequencing Methods. Nitrogenous Bases. Nucleosides. Base linked to a 2-deoxy-D-ribose at 1’ carbon. Nucleotides. Nucleosides with a phosphate at 5’ carbon. Phosphodiester Bond. DNA Polymerase. Determining the Sequence of DNA. Methods:

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DNA Sequencing Methods

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  1. Emily Buckhouse DNA Sequencing Methods

  2. Nitrogenous Bases

  3. Nucleosides • Base linked to a 2-deoxy-D-ribose at 1’ carbon Nucleotides • Nucleosides with a phosphate at 5’ carbon

  4. Phosphodiester Bond • DNA Polymerase

  5. Determining the Sequence of DNA • Methods: • Chain termination or dideoxy method • F. Sanger • Shotgun sequence method • 2nd generation sequence methods • Pyrosequencing

  6. Dideoxy (Sanger) Method • 4 Steps: • Denaturation • Primer attachment and extension of bases • Termination • Gel electrophoresis

  7. Overview: Dideoxy (Sanger) Method 2 3 1 4 Gel electrophoresis 5

  8. Dideoxy (Sanger) Method • ddNTP- 2’,3’-dideoxynucleotide • No 3’ hydroxyl • Terminates chain when incorporated • Add enough so each ddNTP is randomly and completely incorporated at each base

  9. Dideoxy Method • Run four separate reactions each with different ddNTPs • Run on a gel in four separate lanes • Read the gel from the bottom up

  10. Automated Version of the Dideoxy Method

  11. So What’s Wrong With It? • The dideoxy method is good only for 500-750bp reactions • Expensive • Takes a while • The human genome is about 3 billion bp

  12. Human Genome Project • Began in 1990 • Why? • Human evolution • Nature versus nurture • Causes of disease

  13. Shotgun Sequencing • Used to sequence whole genomes • Steps: • DNA is broken up randomly into smaller fragments • Dideoxy method produces reads • Look for overlap of reads

  14. 2nd Generation: Pyrosequencing • Sequencing by synthesis • Advantages: • Accurate • Parallel processing • Easily automated • Eliminates the need for labeled primers and nucleotides • No need for gel electrophoresis

  15. Pyrosequencing • Basic idea: • Visible light is generated and is proportional to the number of incorporated nucleotides • 1pmol DNA = 6*1011 ATP = 6*109 photons at 560nm DNA Polymerase I from E.coli. pyrophospate From fireflies, oxidizes luciferin and generates light

  16. Pyrosequencing • 1st Method • Solid Phase • Immobilized DNA • 3 enzymes • Wash step to remove nucleotides after each addition

  17. Pyrosequencing • 2nd Method • Liquid Phase • 3 enzymes + apyrase (nucleotide degradation enzyme) • Eliminates need for washing step • In the well of a microtiter plate: • primed DNA template • 4 enzymes • Nucleotides are added stepwise • Nucleotide-degrading enzyme degrade previous nucleotides

  18. Pyrosequencing Method:

  19. Pyrosequencing Results:

  20. Pyrosequencing Disadvantages • Smaller sequences • Nonlinear light response after more than 5-6 identical nucleotides

  21. Summary • DNA sequencing is a common procedure • Dideoxy method • Chain termination method • Best for small DNA segments • Whole genome shotgun sequencing • Sequence human genome • Fragments larger DNA strand to manageable chunks • Pyrosequencing • Sequence by synthesis • Accurate and fast

  22. References Applied Biosystems Automated DNA Sequence Chemistry Guide. (2000) Garrett & Grisham. (2007) Biochemistry. Thomson and Brooks/Cole. 3rd ed. Pgs 337-340. Maxam, A. & Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci.74, 560-564. Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res. 11, 3-11. Sanger, F., Nicklen, S., & Coulson, A.R. (1977) DNA Sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.94, 5463-5467. Shendure, J. & Ji, H. (2008) Next-generation DNA Sequencing. Nature Biotech. 26, 1135-1145 Venter, C, et al. (2001) The sequence of the human genome. Science. 291, 1304.

  23. Questions?

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