An algorithm has been developed for the determination of nucleotide sequence from data produced in fluorescence-based automated DNA sequencing instruments employing the four-color strategy. This algorithm takes advantage of object oriented programming techniques for modularity and extensibility. The algorithm is adaptive in that data sets from a wide variety of instruments and sequencing conditions can be used with good results. Confidence values are provided on the base calls as an estimate of accuracy. The algorithm iteratively employs confidence determinations from several different modules, each of which examines a different feature of the data for accurate peak identification. Modules within this system can be added or removed for increased performance or for application to a different task. In comparisons with commercial software, the algorithm performed well.

A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.

We have developed a high speed instrument for automated DNA sequence analysis. The apparatus employs laser excitation and a cooled CCD detector for the parallel detection of up to 18 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (50-100 microns) denaturing polyacrylamide gels. Four hundred and fifty bases of sequence information is obtained from 100 ng of M13 template DNA in less than one hour, corresponding to an overall instrument throughput of over 8000 bases/hr.

Recent work has demonstrated the advantages of ultrathin slab gel electrophoresis for fluorescence-based automated DNA sequence analysis. The increased heat transfer efficiency of the thin (typically 50-100 pm) gels permits higher electric fields to be employed with concomitant increases in separation speed. Issues arise, however, in introducing the laser beam used for fluorescence excitation into the thin gels. This paper presents methods for bringing the excitation beam into the thin gels from the side. This permits a low-power air-cooled aigon ion laser source to be utilized and produces much lower fluorescence and scattering background than alternative approaches. The beam is effectively trapped between the plates due to the high efficiency of reflection at the lowangle grazing incidence of the beam. A theoretical model describing beam throughput was developed which agrees well with experimental observations. In this model, attenuation of the beam intensity is attributed to four factors: aperturing at the entrance of the gel; reflective losses upon entrance into the gel; scattering during transmission through the gel; and reflective losses occurring upon successive "bounces" of the beam from the gelglass interface during propagation of the beam.Electrophoresis in thin gels provides increased heat transfer efficiency, permitting larger electric fields to be employed with correspondingly more rapid separations.* 1-2 This is of particular interest in the area of fluorescence-based automated DNA sequence analysis, where there is a tremendous need for increased throughput from sequencing instruments.3 Kostichka et al. demonstrated an order-of-magnitude increase in separation speed for fluorescence-based DNA sequencing in ultrathin slab gels.4In their work, 18 samples were loaded across an 18 mm width of a 75 pm ultrathin gel, which was cooled from the bottom with a

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