( s wereethanol precipitated dried an...)Automated Fluorescent-Tag Cycle Sequencing

s were ethanol precipitated, dried and resuspended in 6 l of gel loading buffer. Two-microliter aliquots of both sets of reactions were loaded onto a 4.8% Gene Page Plus urea polyacrylamide gel and run on an ABI PRISM 377 DNA sequencer.

Results

figure 2, panel A

figure 2, panel B

The ABI PRISM 377 sequencer automatically scans gels to identify lanes and detect the fluorescent-dye terminators within each lane. The lane tracking was corrected to pass through the center of each lane, where the band fluorescence is maximal. Electropherograms (figure 2, panel A, panel B) were generated for both lanes showing the relative fluorescent intensities of each of the four dideoxy nucleotide reaction terminators (A-Dye Terminator, C-Dye Terminator, G-Dye Terminator and T-Dye Terminator) labeled with a different fluorescent tag. As the excitation laser scans along the electrophoretic axis of the gel, it excites and detects the emission from each tag at each cycle sequencing termination point within the gel. Each of the four fluorescent tags are represented within the electropherogram as different emission line plots, green for A-Dye Terminator, red for T-Dye Terminator, black for G-Dye Terminator and blue for C-Dye Terminator. The four emmision line plots are superimposed to show their spacial relationship within the gel along the electrophoretic axis. The base sequence of the template, as determined by ABI PRISM software, is written above the emission plot lines. The relative number of the nucleotide from the primer is indicated. figure 2 panel A shows the sequencing histogram ge
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