DNA Sequencing at Elevated Temperature by Capillary Electrophoresis

The human genome initiative (
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) has spurred the development of analytical instrumentation for high-throughput
DNA
sequencing. Originally carried out only in poly(acrylamide) slab gels, DNA sequencing is currently to an increasing extent performed in the capillary electrophoresis (CE) format, utilizing narrow bore columns filled with a polymer sieving matrix (
4
). The advantages of the latter technique are the analysis speed, the possibility of online detection and quantitation, as well as the ease of automation, whereas the advantage of the former technique is the parallel capacity of the gel slab. In order to obtain parallel capacity also in the CE format, capillary array electrophoresis (CAE) systems have been developed in recent years (
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), for use in high throughput sequencing applications. Another less obvious feature of the slab-gel systems is that the DNA separation is carried out at temperatures above ambient, due to the heat generated by the relatively large currents (compared to CE) that are present in these systems during electrophoresis (
9
). Elevated temperature has positive effects on the base calling accuracy, the analysis time, and the amount of DNA sequence data that can be obtained from an electrophoretic separation (
10
). In CE, the generally low currents generated and the efficient heat dissipation from the column prevents any appreciable temperature rise during the analysis, so an external heating source must be employed. A number of CE setups that have been designed to operate at elevated temperatures are described in this chapter, along with some fundamental information on DNA sequencing, on the principles of CE, as well as on the DNA structure and the formation of hairpin loops. The effects of elevated temperatures on the polymer separation matrix and on the migration of DNA fragments are discussed as well.