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Biotechnology Processes:

Biotechnology Processes:. DNA Sequencing. Sanger dideoxy method:. a.k.a. Chain Termination Technique How the scientists get the ATCG sequence for the DNA strand!. Animation. Step 1: Create 4 solutions. Each solution contains several single stranded DNA strands created using a PCR.

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Biotechnology Processes:

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  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. 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.

  9. 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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