Cerium Oxide Removes Background Fluorescent Contaminants, 'Green Smears', From Glass Plates Used for Automated Fluorescence-Based DNA Sequencing

摘要

The predominant automated DNA sequencers found in laboratories today use a laser-optics fluorescent dye detection system. In this format, fluorescent dye-labeled DNA fragments are excited by a laser as they electrophorese past a fixed point. The emitted fluorescence is scanned and collected by either a photomultipler tube (PMT) or a charge-coupling device camera (CCD) in the case of the Models 373A and 377 instruments (PE Applied Biosystems, Foster City, CA, USA). One of the problems associated withautomated fluorescence-based DNA sequencing is the appearance of background fluorescence, which can obscure and interfere with signal collection. There appears to be two types of such backgrounds, referred to commonly as a "green smear" One such smear ismobile and migrates with electrophoresis. The second appears fixed and may be either transientor permanent. This second class may be generated by factors such as the general accumulation and deposits of chemicals used in acrylamide gel formation or during plate preparation. In addition, other mechanical problems, impacting gel pouring, may occurfrom a buildup of residues associated with repeated cleaning of glass plates such as carbonates and silicates from tap water, detergents or organic chemicals. Also, we have learned from experience that glass plates may even come from the supplier with preexisting fluorescent backgound contaminants. These contaminants have proven resistant to conventional cleaning methods. The cleanliness and clarity of the glass plates, especially at the signal collection point, is of utmost importance. We have solved this problem by polishing the glass plates with cerium oxide (CeO).