Automated dideoxy DNA sequencing with fluorescent ddNTPs

    Replication of the DNA template strand proceeds with a reaction mixture including the four standard dNTPs and all four ddNTP, each labelled with a different fluorescent dye (ddATP, ddCTP, ddGTP, and ddATP). Random incorporation of the labelled ddNTPs produces a series of DNAfragments in which chain growth has been terminated at each successive position, each one nucleotide longer than the previous. Separation of the fragments by size produces a sequencing ladder as a series of colored bands.

    In an automated DNA sequencer, the fluorescent dye of each band is activated by a scanning laser as it passes a set point at the bottom of the electrophoretic gel. The color of each succesive band is read by a fluorometer, and a computer assembles these as a gel image, which can be read from bottom to top, like a conventional radioactively-labelled sequencing ladder. Multiple sequencing reactions on separate templates are run in parallel: the bands in each ladder are read as a separate electropherogram or chromatogram.

Figure & text © 2010 by Steven M. Carr