SIDE EXCITATION OF FLUORESCENCE IN ULTRATHIN SLAB GEL-ELECTROPHORESIS

Recent work has demonstrated the advantages of ultrathin slab gel elec trophoresis for fluorescence-based automated DNA sequence analysis. Th e increased heat transfer efficiency of the thin (typically 50-100 mu m) 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. Th is paper presents methods for bringing the excitation beam into the th in gels from the side. This permits a low-power air-cooled argon ion l aser source to be utilized and produces much lower fluorescence and sc attering background than alternative approaches. The beam is effective ly trapped between the plates due to the high efficiency of reflection at the low-angle grazing incidence of the beam. A theoretical model d escribing beam throughput was developed which agrees web with experime ntal observations. In this model, attenuation of the beam intensity is attributed to four factors: aperturing at the entrance of the gel; re flective losses upon entrance into the gel; scattering during transmis sion through the gel; and reflective losses occurring upon successive ''bounces'' of the beam from the gel-glass interface during propagatio n of the beam.