Automated Sequencing

• An improvement to manual sequencing.

  Automated Sequencing
• Uses double-stranded templates and cycle sequencing that doesn’t require sequential addition of reagents to start and stop the reaction.

  Automated Sequencing
• In automated sequencing, distinct colors (peak wavelengths) of fluorescence emission pertains to the appropriate nucleotide and is the basis of the read instead of the lane.
• Fluorescent dyes are covalently attached to each of the ddNTPs instead of the primer.


  Dye Terminator
• All four sequencing reactions are performed in the same tube.

  Dye Terminator
• Product fragments are labeled at the 3’ end.

  Dye Terminator
• Four different fluorescent dyes are attached to four separate aliquotes of the primer.

  Dye Primer
• Products are labeled at 5’ end with the dye color associated to the ddNTP at the end of the fragment.

  Dye Primer
• Procedure of Automated Sequencing.

  1. Preparation of Ladder
  2. Electrophoresis
  3. Sequence Interpretation
• Procedure: After the sequencing reaction, excess terminators are removed.

  Preparation of Ladder.
• Procedure: Clean with columns or beads or by ethanol precipitation.

  Preparation of Ladder
• Procedure: Denaturation of Fragments

  Preparation of Ladder
• Procedure: Ran on the same lane, avoiding lane-to-lane migration variations.

  Electrophoresis
• Procedure: Migrating fragments pass a laser beam and then an detector.

  Electrophoresis
• Procedure: Detectors convert fluorescence to an electrical signal that is recognized by computer software or as a flash of peak of color.

  Electrophoresis
• Procedure: Reading of Sequence

  Sequence Interpretation
• Procedure: Bright flashes of fluorescence due to failure to clean the ladder.

  Sequence Interpretation
• Procedure: Poor starting material leads to poor quality sequence.

  Sequence Interpretation