TRITYL MONITORING OF AUTOMATED DNA SYNTHESIZER OPERATION BY CONDUCTIVITY - A NEW METHOD OF REAL-TIME ANALYSIS

AutoAnalysis is a new method for detecting and quantitating the trityl cation released each cycle on automated DNA synthesizers. The trityl (dimethoxytrityl) cation is removed from the growing oligonucleotide a fter each base addition and is a useful measure of synthesis efficienc y. The traditional absorbance method of collecting each trityl effluen t with a fraction collector, followed hy dilution with an acid solutio n and careful quantitation by UV/VIS spectroscopy is costly, tedious a nd prone to error. The absorbance method for trityl cation analysis mu st usually wait until the synthesis is complete. Interruption of a fai led operation, for a variety of reasons, such as an empty reagent rese rvoir, is thus not possible. Taking advantage of the conductive proper ties of the trityl cation, immediate and real-time quantitation is now possible by integrating the total conductance of the flowing stream d uring the detritylation step after each nucleoside addition in DNA syn thesis. A conductivity cell is mounted downstream, past the synthesis column. The conductivity signal is processed and displayed as the curr ent average stepwise yield and overall yield. If the yield drops below a pre-set threshold value because of a failure situation, the synthes izer will interrupt, preserving reagents. AutoAnalysis allows trityl m onitoring with complete automation on the Applied Biosystems Models 39 2 and 394 DNA/RNA Synthesizers.