How Sanger Sequencing Works? (Classic Sanger Method)

Learn the definition of DNA sequencing and the historical background of the Sanger method developed by Frederick Sanger in 1977. Understand that this technique uses in vitro DNA synthesis based on principles of DNA replication.

Review the four essential components needed for DNA synthesis: DNA template strand, primer, deoxynucleotides (dNTPs), and DNA polymerase. Explain the role of the 3-prime hydroxyl group from the primer in initiating phosphodiester bond formation.

Demonstrate the chemical structure difference between normal deoxynucleotides and dideoxy nucleotides (ddNTPs), showing the absence of the 3-prime hydroxyl group in ddNTPs. Explain how incorporation of ddNTPs terminates DNA synthesis, forming the basis of the chain termination method.

Prepare four separate reaction mixtures, each containing DNA template, primer, DNA polymerase, and all four standard dNTPs in large amounts. Add a different ddNTP (ddATP, ddCTP, ddGTP, or ddTTP) in small amounts to each reaction to create chain termination variants.

Allow DNA polymerase to synthesize new strands in all four reactions. Whenever a ddNTP is randomly incorporated instead of the normal dNTP, synthesis terminates, producing a collection of partial DNA fragments of different lengths, each ending at a different position.

Load the contents of each reaction into separate lanes of a polyacrylamide gel and run electrophoresis. Fragments migrate based on size, with smallest fragments traveling farthest and largest fragments traveling shortest distance.

Produce an autoradiograph of the gel using the radioactive labels on primers. Read the sequence from bottom to top according to increasing fragment size, matching each band position to its corresponding ddNTP lane (A, T, G, or C).

Convert the newly synthesized strand sequence read from the autoradiograph into the complementary template strand sequence. Confirm the directionality is 5-prime to 3-prime, successfully obtaining the complete DNA sequence.