Product Options Increase In DNA Sequencing Arena

DNA sequence analysis is one of the pivotal methods of modern molecular biology. It plays a central role in virtually every project that involves the cloning, characterization, and manipulation of RNA or DNA. Since the development of rapid sequencing techniques in the early 1970s, more than 20 million bases have been sequenced by manual techniques. This DNA sequencing has all been accomplished with either of two techniques: the enzymatic method of sequencing, developed by Sanger and Coulson, and the chemical method, developed by Maxam and Gilbert. In both methods, as they are conventionally practiced, sets of radiolabeled DNA fragments with a common origin, but terminating at a particular base or bases, are produced in the sequencmg reactions. These fragment sets are loaded in adjacent lanes of a high-resolution polyacrylamide slab gel separated by electrophoresis, and a film image is obtained by autoradiography. Analysis of the autoradiogram yields the DNA sequence of the unknown region. In the last three years, dramatic changes have occurred in the technology of DNA sequencing. Several instruments have become available that automate to varying degrees aspects of DNA sequencing (see the accompanying chart). These instruments fall into three major categories, differing primarily in the nature of the detection of the DNA fragments. The instruments most closely related to conventional sequence analysis are those in which electrophoresis and autoradiography are still performed in the conventional manner, but the autoradiogram analysis is automated or semiautomated. These instruments require the least change for users from the procedures with which they are accustomed. The instruments can reduce the time required for sequence analysis, a procedure that used to force scientists to spend long periods hunched over a light box. A logical extrapolation of this approach is to devise systems in which the radioactive DNA bands are detected directly within the gel, either during or after electrophoresis. These systems eliminate the need for autoradiography, replacing the use of film for detection with the use of detectors similar to those used in scintillation counting. Computerized analysis of the acquired data to automatically yield the DNA sequence may further reduce the labor of sequencing. The third class of instruments are those in which radioactivitybased detection is replaced with fluorescence-based detection. In this method, thus far applied only to the enzymatic method of sequencing, fluorescent tags are covalently attached to the DNA molecules being analyzed, and are detected within the gel using very sensitive laser-based detection systems. In one approach, the tags are attached to the primer DNA fragment used to initiate strand synthesis in sequencing by the enzymatic method, alternatively, the tags may be attached to the chain-terminating nucleosides. In either case, multiple fluorophores may be employed—one for each of the four sequencing reactions—and the four reactions may be coelectrophoresed in a single gel lane. In this case, the sequence is determined by the temporal order in which the different fluorophores pass through the detector, rather than by their spatial pattern. This strategy increases the throughput of the instruments, and decreases the difficulty of achieving an accurate analysis of the data. These instruments are ushering in a new age of molecular biology. In this era, skilled scientists will no longer need to perform tedious, repetitive procedures to obtain the data essential for progress, but may rather use automated instruments and computer technologies to acquire such information. They will’ thus be able to focus their attention instead on the study of the fundamental biology and chemistry of living systems. Lloyd Smith is an assistant professor in the department of chemistry at the University of Wisconsin, Madison.

Product Options Increase In DNA Sequencing Arena

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