Biomedical Engineering Reference
In-Depth Information
First-Generation Automated Sanger DNA Sequencing
Toward the end of the manual-sequencing era, in 1986 the fi rst generation of “auto-
mated sequencers,” pioneered by Applied BioSystems (now part of Life
Technologies), appeared and included automation of the gel electrophoresis steps,
detection of the fl uorescent DNA band patterns, and analysis of bands (Fig. 1.1 ;
Smith et al. 1986 ). The four sets of sequencing reactions were loaded onto slab gels
manually and electrophoresis automated. The sequence was obtained by recording
the moving bands sequentially past a detector at the bottom of the gel. The replace-
ment of radioactivity with fl uorescently labeled primers or ddNTPs and develop-
ment of polymerases that could effectively incorporate these dyes were essential to
the automation process and this remains the general method of choice for these
fi rst-generation automated DNA sequencers. Today, automated DNA sequencing
performed today makes use of automated capillary electrophoresis, which typically
analyzes 8-96 sequencing reactions simultaneously, in combination with the use of
the latest generation of fl uorescent dyes that exhibit strong and distinct fl uorescent
emissions (Fig. 1.1 ; Ju et al. 1996 ). Thus, implementations of “fi rst-generation auto-
mated DNA sequencing” had key advantages over methods described for the origi-
nal Sanger sequencing, namely, the elimination of radioactivity use, “one-lane”
sequencing, “one-tube” reactions, automated base calling, and elimination of slab-
gel technology in favor of multi-capillary electrophoresis with automatic,
electrokinetic-injection lane loading.
Automated Sequencing Factories
Craig Venter and colleagues at NIH used the ABI 370A DNA sequencer to deter-
mine the sequence of a gene (Gocayne et al. 1987 ). At NIH, Venter set up a sequenc-
ing facility containing six automated sequencers and two ABI Catalyst robots. In
1992, Venter established The Institute for Genomic Research (TIGR) to expand his
sequencing operation with 30 ABI 373A automated sequencers and 17 ABI Catalyst
800 robots (Adams et al. 1994 ). This was a real factory with teams dedicated to dif-
ferent steps in the sequencing process such as template preparation, gel pouring,
and sequencer operation. Data analysis was integrated into the process so that prob-
lems in earlier steps could be detected and corrected as soon as possible.
An early demonstration of the power of automated sequencing was the develop-
ment of the expressed sequence tag (EST) approach to gene discovery. In this
approach, cDNA copies of messenger RNA were cloned at random and subjected to
automated sequencing. In the fi rst report from Venter and colleagues in 1991, 337
new human genes were reported, 48 homologous to genes from other organisms
(Adams et al. 1991 ). This approach was adopted by many genome projects. Today,
the EST database contains over 43 million ESTs from over 1,300 different organ-
isms. Another early application of the automated sequencer was the worm genome
sequencing project which was underway by 1992 with the beginning elements of a
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