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Biotechnology Processes:

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Biotechnology Processes: DNA Sequencing Sanger dideoxy method: a.k.a. Chain Termination Technique How the scientists get the ATCG sequence for the DNA strand! – PowerPoint PPT presentation

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Title: Biotechnology Processes:


1
Biotechnology Processes
  • DNA Sequencing

2
Sanger dideoxy method
  • a.k.a. Chain Termination Technique
  • How the scientists get the ATCG sequence for the
    DNA strand!

3
Animation
4
Step 1 Create 4 solutions
  • Each solution contains several single stranded
    DNA strands created using a PCR.
  • Each solution contains DNA polymerase and a
    primer
  • Each solution contains high concentrations of all
    of the 4 nucleotides, A, C, G, and T (in
    deoxynucleoside triphosphate form).

5
Step 1 Continued
  • Each solution contains 1 of the 4 nitrogen base
    pairs (either A, G, T or C) that has been altered
    to be a radioactively labelled dideoxy-
    nucleosides triphosphate

6
Dideoxynucleoside Triphosphates
  • A nucleotide in which a second oxygen has been
    removed from the ribose sugar
  • 1 removal of oxygen gives Deoxyribose, 2 removals
    of oxygen gives Dideoxyribose!
  • Dideoxynucleotides are named ddATP, ddCTP, ddGTP
    and ddTTP

7
Step 2 Replication
  • Allow the DNA to replicate, starting from the
    primer, forming complimentary strands
  • Since solution contains regular deoxynucleosides
    as they are incorporated the chain elongation
    continues,
  • BUT
  • Since -OH is missing from dideoxy-nucleosides
    they end transcription (nothing to bind to)
  • Copied fragments end up being different lengths,
    depending on where the dd-nucleosides were
    randomly added!

8
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9
Step 3 Creating an Autoradiogram
  • The DNA fragments are then loaded onto a
    polyacrylamide gel tray.
  • Each container in one of 4 runs.
  • Each of the radioactive forms of the nitrogen
    base are placed in a different well.
  • Placed against X-ray form.

10
  • Sequence of DNA read from autoradiogram.
  • read from positive end to negative (bottom to
    top)
  • Gives a 3' to 5' COMPLIMENTARY strand
  • To get original strand, must find the
    complimentary bases to the decoded strand and
    reverse order, to get in 5' to 3' direction...

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