• This method of DNA sequencing is based on the classic chain termination method developed by Frederick Sanger, and uses the polymerase chain reaction (PCR).
  • Like any PCR method (seeĀ here), this sequencing technique involves repeated cycles of denaturation, annealing of primers and extension using a high temperature polymerase enzyme. How it differs from conventional PCR is in the use of labeled versions of the dideoxyribonucleotide triphosphates (ddNTPs) in addition to unlabelled ones.
  • These modified ddNTPs are incorporated into the PCR products made, however they also stop the extension of the chain (hence they are called terminators).The use of these terminating ddNTPs creates a selection of DNA fragments of differing size, each of which ends with a particular nucleotide which is labeled with a different coloured dye.
  • The fragments can then be separated according to size. In conventional agarose gel electrophoresis, a sample of DNA is loaded into a well in the agarose and an electric current applied. Because the conditions within the gel give the DNA an overall negative charge, the electric field pushes the DNA through the gel from the negative terminal to the positive terminal. Smaller fragments of DNA can move more quickly through the gel than larger fragments, and so the DNA separates out into regions of the gel which contain fragments of a similar size. If a dye which binds to DNA is incorporated into the agarose (eg. ethidium bromide or SYBR-Green), these regions can be visualized as bands. The bands located further down the gel (ie. closer to the positive terminal) represent DNA fragments of a smaller size than those located closer to the negative terminal.
  • Dye terminator sequencing can be performed using a conventional gel. However in more modern automated systems, the electrophoresis is performed in a thin tube called a capillary. No dye needs to be incorporated into the gel as the DNA fragments are already fluorescently labeled using the dye terminators.
  • As the soup of differently sized DNA fragments separates out in the capillary, it produces a series of coloured bands. Each band represents fragments of DNA of a particular size, and each colour represents the base at which the fragment terminates. The shorter fragments, representing the bases at the beginning of the sequence will move through the capillary first.
  • An excitation laser shines through the capillary and the light emitted by the fluorescent dye is it returns to a lower energy level is detected by a detector system. As each coloured band is detected, it creates a signal which is processed by the sequencer and presented as a peak on a graph. Each peak represents a different base

Use of PCR and Dye Terminators to create lengths of DNA which end in particular labelled nucleotides

Separation of lengths of DNA with labeled terminal nucleotides through a capillary