The Colors that Do Magic
How Fluorescence Enabled Automation in Nucleic Acid Sequencing
Weill Cornell Medicine–Qatar
Weill Cornell Medicine–Qatar
Weill Cornell Medical College–Qatar
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.
- 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.
KeywordsSequencing; dideoxyribonucleotide; DNA polymerase; fluorescence; Sanger; chromatogram; DNA synthesis; agarose gel; nucleotides; dNTP; ddNTP
Educational LevelHigh school, Undergraduate lower division, Undergraduate upper division
Type / MethodsDirected, Laboratory
Subject HeadingsBiology (General) | Molecular Biology | Bioinformatics |
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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)
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The following video(s) are recommended for use in association with this case study.
- DNA Sequencing: The Chain Termination Method (Sanger Method)
Students are directed to watch this brief animation explaining the Sanger method of DNA sequencing in the first section of this case study. Running time: 3:00 min. Produced by Conor, 2012.