Autorad

DNA sequencing autoradiogram from SM Carr lab, 1993

    Six different DNA samples are examined with four radioisotope labeling 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 by reading 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 read 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.