Sanger Sequencing Explained: The Original Method to Modern DNA Sequencing

Learn the structural differences between normal nucleotides (dNTPs) and chain-terminating nucleotides (ddNTPs). Understand how ddNTPs lack a 3' hydroxyl group, preventing further nucleotide addition and terminating DNA synthesis.

Set up four separate reaction tubes, one for each ddNTP type (ddATP, ddGTP, ddCTP, ddTTP). Add primer, DNA template, DNA polymerase, all four normal dNTPs, and one radioactively-labeled ddNTP to each tube.

Heat the DNA mixture to 100°C to denature double strands into single strands. Cool to 67°C to allow sequencing primers to bind. Add DNA polymerase to extend the template until a ddNTP randomly incorporates and terminates the chain.

Mix the four reaction tubes with loading dye and load each into separate lanes of a polyacrylamide gel. Run electrophoresis to separate fragments by size, then dry the gel onto paper and detect radioactive signals using X-ray film to reveal bands.

Adopt Applied Biosystems AB370A sequencer with fluorescent dyes replacing radioactive labels and automated laser detection. Mix all four reactions in a single gel lane and use computer-aided base calling, enabling 16 samples per gel with 450 nucleotide read length.

Incorporate Taq polymerase into Sanger sequencing to enable thermal cycling similar to PCR. Use repeated cycles of DNA melting, primer annealing, and extension to amplify fragments linearly, increasing fluorescent signal and reducing DNA requirements.

Transition from slab PAGE gels to capillary electrophoresis using the ABI PRISM 310 system launched in 1995. Load samples in 96-well plates for automated analysis with single-capillary separation, achieving 600 base pair reads in under 3 hours per sample.

Deploy the ABI PRISM 3700 sequencer with 96 capillaries, processing 1,536 samples daily with 800 base pair reads per sample. Use this technology to successfully sequence the human genome through both the public Human Genome Project and private Celera Genomics.