Dideoxy sequencing

Gel images from an automated DNA sequencer

    From the original DNA template, the dideoxy sequencing reaction synthesizes a new DNA strand that incorporates a set of fluorescently-labeled ddNTPs into the DNA (left). The newly synthesized DNA is separated by size in an electrophoretic field at high voltage, and the fluorescence colours are activated by a scanning laser, tracked, and read by a photometer. The fluorescent dyes used are four slightly different wavelengths of green: the gel image (middle) is a "false colour" representation, in which these wavelengths are shown as distinct A C G & T. Each of 24 different DNA sequences is a separate sequencing reaction; automatic tracking for one lane is shown by the white trace.  In the magnified view (right), the sequencing ladder in each lane has four different coloured bands that correspond to each of four DNA bases.  Each channel is converted to an individual chromatogram for that DNA sequence; the order of coloured peaks is converted to a DNA sequence. For example, the first few bases of the fourth lane boxed in red will be read as ATTTGAATTC .

    Because the four bases appears as different colors, all four reactions for any sample can be run in and read in a single lane, rather than four when a 32P label is used. Automated DNA sequencing revolutionized the field and made possible the Human Genome Project. The 24 reactions show here cover the complete 16 Kbp sequence of a human mtDNA genome. The most advanced machine of this time could run 96 samples simultaneously.  [Click here for an animation].

HOMEWORK: Popular accounts like the movie "Jurassic Park" suggest that "DNA sequences are read by a laser'" Explain why this is incorrect.

Images & text material © 2024 by Steven M. Carr