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The Colors that Do Magic

How Fluorescence Enabled Automation in Nucleic Acid Sequencing


Author(s)

Ghizlane Bendriss
Premedical Division
Weill Cornell Medicine–Qatar
ghb2002@qatar-med.cornell.edu
Ali Chaari
Premedical Division
Weill Cornell Medicine–Qatar
alc2033@qatar-med.cornell.edu
Kuei-Chiu Chen
Premedical Division
Weill Cornell Medical College–Qatar
kuc2005@qatar-med.cornell.edu

Abstract

This directed case study with a lab component tells the story of “Sara,” a college sophomore majoring in biological science. Sara has been awarded an internship in a lab focusing on sequencing gene markers for species identification. On her first day, she learns that the sequencing machine is temporarily out of service, so she is asked to prepare the reaction tube for sequencing the following day using the traditional Sanger method. Sara experiences experimental errors that lead to unexpected results, but her good lab notes allow her to pinpoint the cause. When she runs her prepared reaction tube the following day through the repaired automated sequencing machine she realizes that the invention of Professor Leroy Hood at Caltech and the team of DuPont Research Station has paved the way for second, third, and next generation sequencing (NGS). Central to the case is an activity in which students simulate the synthesizing of new strands of DNA by playing the role of DNA polymerase using the labeled dideoxynucleotide sequencing method.  Originally designed for a first-year medical college general biology class, this case can also be used in high school and college-level biology or biochemistry courses.


Objectives

  • Describe the principles of Sanger sequencing.
  • Simulate the DNA synthesis process
  • Describe the reactants needed for sequencing and their function
  • Visualize the actions taking place in a sequencing reaction tube.
  • Differentiate the 3´ and 5´ ends of a sequence in a replication process.
  • Determine the sequence of a DNA template from an agarose gel result and from a chromatogram.
  • Describe the advantage of using fluorescent labels on the primer in a sequencing reaction as developed in Caltech.
  • Describe the advantage of using fluorescent labels on ddNTPs in a sequencing reaction as developed by the DuPont group.
  • Troubleshoot sequencing experiments.

Keywords

Sequencing; dideoxyribonucleotide; DNA polymerase; fluorescence; Sanger; chromatogram; DNA synthesis; agarose gel; nucleotides; dNTP; ddNTP

Topical Areas

N/A

Educational Level

High school, Undergraduate lower division, Undergraduate upper division

Format

PDF, PowerPoint

Type / Methods

Directed, Laboratory

Language

English

Subject Headings

Biology (General)  |   Molecular Biology  |   Bioinformatics  |  


Date Posted

08/23/2021

Teaching Notes

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Supplemental Materials

As detailed in the teaching notes, this case study requires the instructor to prepare the materials included in the PowerPoint presentation below.

  
  sanger_sequencing_sup.pptx (~269 KB)

Answer Key

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Videos

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