Sanger sequencing

Describe the three components of nucleotides (nitrogenous base, sugar residue, phosphate group) and explain how removing the hydroxyl group at the 3' end of the sugar creates dideoxynucleotides, which cannot form phosphodiester bonds.

Divide template DNA into four separate tubes, each containing primer, all four standard dNTPs, and DNA polymerase enzyme. Add a single radio-labeled dideoxynucleotide at low concentration to each tube (ddATP, ddTTP, ddGTP, or ddCTP).

Allow DNA polymerase to add dNTPs to the template DNA. When a dideoxynucleotide is randomly incorporated, the reaction stops due to the absence of the 3' hydroxyl group, producing fragments of varying lengths with terminated chains.

Perform polyacrylamide gel electrophoresis and autoradiography to separate the radio-labeled DNA fragments by length. The position of each band corresponds to the location of a specific nucleotide in the sequence.

Read the autoradiogram from bottom to top, where each band represents a terminated fragment. The sequence of bands reveals the order of nucleotides in the original DNA template.

Label each dideoxynucleotide (ddATP, ddTTP, ddGTP, ddCTP) with a distinct fluorescent dye (yellow, green, blue, or red) for modern Sanger sequencing. This enables all reactions to occur in a single tube instead of four separate tubes.

Run polyacrylamide gel electrophoresis or capillary electrophoresis to separate the fluorescently-labeled chain-terminated DNA fragments. Use a UV light detector or charge-coupled device (CCD) to detect the fluorescent signals from each fragment.