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Targeted Amplicon Genotyping by Sanger Sequencing
Anthony Torres1, Reginald Gaudino2,1
1Cannabis Research Institute, Discovery Partners Institute, University of Illinois System, Chicago, IL, USA.
Methods in Molecular Biology (Clifton, N.J.)
|June 28, 2025
Summary
Sanger sequencing is a cost-effective, accurate method for targeted DNA sequencing. This protocol details primer design, chain termination, electrophoresis, and chromatogram analysis for identifying genetic variations.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Sanger sequencing remains a valuable tool for targeted DNA analysis.
- Conventional methods offer a balance of speed, cost-effectiveness, and accuracy.
Purpose of the Study:
- To present a detailed protocol for performing Sanger sequencing.
- To outline the steps for accurate base identification and variation detection.
Main Methods:
- Designing specific primers for DNA amplification.
- Incorporating fluorophore-labeled, chain-terminating dideoxynucleotides.
- Separating DNA fragments via electrophoresis and analyzing chromatograms.
Main Results:
- Precise base identification within DNA amplicons.
- Detection of nucleotide changes indicative of genetic variations.
- Facilitation of amino acid variation analysis in targeted genes.
Conclusions:
- Sanger sequencing provides a reliable method for targeted genetic analysis.
- The protocol enables accurate identification of nucleotide and amino acid variations.
- This technique remains relevant for cost-effective and rapid genetic studies.
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