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Automated Sequencer

The Need For Automated Sequencing

When chain termination sequencing is performed manually, each of four reaction tubes contains a different type of terminator base as well as a radioactive nucleotide for labeling the DNA fragments as they are made. Each of the four reactions is electrophoresed in a separate lane of a gel, and X-ray film is used to detect the fragments. Using this technique, a dedicated and skilled technician can determine the sequence of as many as 5,000 bases in a week. Demand for the ability to read more sequence in a shorter amount of time, however, led to the development of instruments that could, with the aid of computers, automate the DNA sequencing process.

The first step toward this goal was achieved in 1985, when Leroy Hood at the California Institute of Technology attached fluorescent dyes to the primer used in the sequencing reactions; each different color dye (blue, green, yellow, and red) was matched with a different terminator base. He and Michael Hunkapiller from Applied Biosystems, Inc. (ABI) built an instrument, dubbed the ABI Model 370, to read the sequence of the dyelabeled fragments. It was equipped with an argon ion laser for exciting the dyes, a flat gel laid between two glass plates (referred to as a "slab" gel) capable of sixteen-lane electrophoresis, and a Hewlett-Packard Vectra computer boasting 640 megabytes of memory for data analysis.

Using fluorescent dyes, all four sequencing reactions could now be loaded into a single gel lane. As the fragments electrophoresed, the beam of the laser focused at the bottom of the gel made the dye-labeled fragments glow as they passed. The color of each dye-labeled fragment was then interpreted by the computer as a specific base (A if green, C if blue, G if yellow, and T if red). Over 350 bases could be read per lane. With this new automated approach, a technician could read more sequence in a day than could be read manually in an entire week.

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Medicine EncyclopediaGenetics in Medicine - Part 1Automated Sequencer - The Need For Automated Sequencing, Refinements In Automation