Biotechnology%20Processes:

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Biotechnology Processes

• DNA Sequencing

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Sanger dideoxy method

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

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Animation
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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).

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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

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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

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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!

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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.

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• 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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