MULTI-FACILITY SURVEY OF OLIGONUCLEOTIDE SYNTHESIS AND AN EXAMINATIONOF THE PERFORMANCE OF UNPURIFIED PRIMERS IN AUTOMATED DNA-SEQUENCING

The purity of 208 crude synthetic 25- and 50-base oligonucleotides syn thesized in 71 DNA core facilities was assessed by capillary electroph oresis (CE), and the average coupling efficiency of each synthesis was determined. The median average coupling efficiencies of the 25-mers a nd 50-mers were 98.9% and 98.7%, respectively, and 85% of the samples exceeded the minimum industry standard of 98% average coupling efficie ncy. The overall yields estimated by on-line trityl monitors showed po or agreement with the empirically determined yield, and accuracy of th e monitors decreased as synthesis efficiency decreased. The performanc e of the unpurified 25-base oligonucleotides, ranging in purity from 1 4% to 94%, as primers for automated DNA sequencing was evaluated. Over 85% of these oligonucleotides exhibited an unedited sequencing accura cy of >97.5% over the 400-base test sequence. Surprisingly, sequencing performance was not strictly related to primer purity, though a marke d loss of performance was observed for primers less than or equal to 7 0% pure (less than or equal to 98.5% coupling efficiency). Thus, the v ast majority of the oligonucleotides synthesized by the 71 core facili ties participating in this study were of high quality and performed we ll as sequencing primers without post-synthesis purification or desalt ing. Finally, our results suggest that an increase in the standard min imum performance specifications of DNA synthesis instruments and reage nts from greater than or equal to 98% to greater than or equal to 98.5 % average coupling efficiency, or the development of rapid, inexpensiv e and efficient methods to detect syntheses below the 98.5% threshold, could obviate post-synthesis purification of sequencing primers.