DNA sequencing autoradiogram
from SM Carr lab, ca. 1993
Six different DNA samples
are
examined with four
dideoxy (ddN) sequencing
reactions each, using respectively ddA,
ddG, ddC, & ddT. The dideoxy bases are
each tagged with 32P, the reactions
separated in an acrylamide gel at high voltage, the gel dried onto
filter paper, and overlain with X-ray film for several hours. The
lanes for each individual
are read from
left to right as GATC,
from
bottom to top. Note that bands at the bottom of the gel are
initially
widely spaced, and become more compressed towards the top. The
shortest DNA fragments
are found at the bottom: because the difference between fragments
of
length 50 b vs 51 b is 2%, the mobility difference is easily
detected. Towards the top, the difference between 400b vs 401b is
0.25%, and the mobility difference is correspondingly smaller. In
practice, two electrophoresis runs are made, one for 1.5
hrs and a second for 4 hrs: the first separates short fragments,
the
latter runs these fragments off the gel but allows separation of
the
larger fragments.
Other artifacts observable in the gel include
(1) weak reactions [first
sample, lane 1], (2) small imperfections in the gel (e.g.,
bubbles)
that distort even migration [third sample, lane 1], and (3) the "smiley
face", in which reactions near the edges of the gel tend to
run faster and curve upward [first sample].
Homework:
Starting
at
the the dark double bars about a third of the way up the
gel, read the sequence of
each
of the six DNAs. Do any
of the
individuals have different DNA
sequences from each other?
