DNA sequencing autoradiogram
from SM Carr lab, ca. 1993
Six different DNA samples are examined
with four
sequencing reactions each. The lanes for each individual are read
from
left to right as GATC,
and in the 5'
3' direction from
bottom to top. Note that bands at the bottom are
well-spaced but blurry, bands in the middle are well-spaced and
sharp, and bands at the top are sharp but closely-packed so as to
become increasingly difficult to read. An experience reader could
read ca. 400b from this gel. Two electrophoresis runs, of 1.5 and
4 hrs, are sufficient to resolve ca. 800 bases. If the complementary
strand is also sequenced, in two runs, combined sequences of
> 1,000 bases can be compiled from the four runs.
[Bio2250
labs were done with autorads
like this, before the advent of
automated sequencing].
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. Identify the sequence differences
among individuals.
Advanced
Homework: Reading the gel below gives the 5'3' sequence of the "forward"
or sense strand. Reading the gel sequence the "reverse"
or partner strand will also give a 5'3'
sequence. To align these sequences, it is necessary to use a
computer to generate the reverse complement sequence from the
partner strand. This makes it difficult to check for reading
differences between the two experiments. Devise a (clever) method for reading the
reverse complementary sequence of the partner strand
directly from the gel, so that it aligns directly
with the sense strand.
Figure & text
material © 2009 by
Steven M. Carr; not to be
reproduced without permission