Sanger Sequencing | Animated Biology

Remove ONE oxygen from a nucleotide… and suddenly you can read DNA letter by letter. That's the trick that earned Frederick Sanger his second Nobel Prize in Chemistry. In this video we go through it step by step, fully animated: how DNA is denatured, how the primer anneals and the polymerase copies; why the OH on the 3′ carbon is the key to everything; how a ddNTP stops the chain; how it was done in 1977 with four reactions, a gel and radioactivity; how it's done today with fluorescence and capillary electrophoresis; and what it's still used for in the era of next-generation sequencing. The chemical structures in the video (the dNTP and the ddNTP) are drawn from their real formulas in PubChem, not redrawn by hand. ⏱️ CHAPTERS 0:00 Intro 0:44 1977: Sanger's idea 1:07 Denaturing and annealing the primer 1:35 The polymerase copies the template 2:14 The 3′ OH: how DNA grows 2:45 The ddNTP: the nucleotide that stops the chain 3:28 Sanger's trick 4:07 The four reactions of 1977 4:44 Gel electrophoresis 5:15 Autoradiography: reading DNA with radioactivity 5:44 Fluorescence: one color per base 6:10 Capillary electrophoresis and the chromatogram 6:45 Multi-capillary sequencers 7:10 Sanger today: the gold standard 7:27 Heterozygotes: two peaks in one spot 7:50 Is it still necessary? 📄 THE PAPERS The original paper on the method (December 1977): Sanger F., Nicklen S., Coulson A.R. (1977). "DNA sequencing with chain-terminating inhibitors". PNAS. https://doi.org/10.1073/pnas.74.12.5463 The complete genome of phage φX174, finished with the new method: Sanger F., Coulson A.R., Friedmann T. et al. (1978). "The nucleotide sequence of bacteriophage φX174". Journal of Molecular Biology. https://doi.org/10.1016/0022-2836(78)... The gold standard in the clinical lab: Rehm H.L., Bale S.J., Bayrak-Toydemir P. et al. (2013). "ACMG clinical laboratory standards for next-generation sequencing". Genetics in Medicine. https://doi.org/10.1038/gim.2013.92 Does everything still need Sanger confirmation? Two data-driven answers: Beck T.F., Mullikin J.C. et al. (2016). "Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants". Clinical Chemistry. https://doi.org/10.1373/clinchem.2015... Arteche-López A., Ávila-Fernández A., Romero R. et al. (2021). "Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes". Scientific Reports. (Open access.) https://doi.org/10.1038/s41598-021-85... Heterozygotes in the chromatogram: Korch C., Drabkin H. (1999). "Improved DNA Sequencing Accuracy and Detection of Heterozygous Alleles Using Manganese Citrate and Different Fluorescent Dye Terminators". Genome Research. https://doi.org/10.1101/gr.9.6.588 The special letters (R, Y…) for ambiguous bases: Cornish-Bowden A. (1985). "Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984". Nucleic Acids Research. https://doi.org/10.1093/nar/13.9.3021 Found a mistake, or is there a topic you'd like me to explain? The comments are yours.