Fig. 12. DNA Sequencing.

Dideoxy sequencing (also called chain- termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the diagram contains

• a DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a new strand of DNA at the point where the primer is hybridized to the template;

• the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the DNA strand;

• one labeled deoxynucleotide triphosphate (using a radioactive element or dye); and

• one dideoxynucleotide triphosphate, which terminates the growing chain wherever it is incorporated. Tube A has didATP, tube C has didCTP, etc.

For example, in the A reaction tube the ratio of the dATP to didATP is adjusted so that each tube will have a collection of DNA fragments with a didATP incorporated for each adenine position on the template DNA fragments. The fragments of varying length are then separated by electrophoresis (1) and the positions of the nucleotides analyzed to determine sequence. The fragments are separated on the basis of size, with the shorter fragments moving faster and appearing at the bottom of the gel. Sequence is read from bottom to top (2). (Source: see Fig. 11.)

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