Multi-facility survey of oligonucleotide synthesis and an examination of the performance of unpurified primers in automated DNA sequencing.
The purity of 208 crude synthetic 25- and 50-base oligonucleotides synthesized in 71 DNA core facilities was assessed by capillary electrophoresis (CE), and the average coupling efficiency of each synthesis was determined. The median average coupling efficiencies of the 25-mers and 50-mers were 98.9% and 98.7%, respectively, and 85% of the samples exceeded the minimum industry standard of 98% average coupling efficiency. The overall yields estimated by on-line trityl monitors showed poor agreement with the empirically determined yield, and accuracy of the monitors decreased as synthesis efficiency decreased. The performance of the unpurified 25-base oligonucleotides, ranging in purity from 14% to 94%, as primers for automated DNA sequencing was evaluated. Over 85% of these oligonucleotides exhibited an unedited sequencing accuracy of > 97.5% over the 400-base test sequence. Surprisingly, sequencing performance was not strictly related to primer purity, though a marked loss of performance was observed for primers < or = 70% pure (< or = 98.5% coupling efficiency). Thus, the vast majority of the oligonucleotides synthesized by the 71 core facilities participating in this study were of high quality and performed well as sequencing primers without post-synthesis purification or desalting. Finally, our results suggest that an increase in the standard minimum performance specifications of DNA synthesis instruments and reagents from > or = 98% to > or = 98.5% average coupling efficiency, or the development of rapid, inexpensive and efficient methods to detect syntheses below the 98.5% threshold, could obviate post synthesis purification of sequencing primers.
University Core DNA Services, University of Calgary, AB Canada. rtpon@acs.ucalgary.ca
R T
RT
Pon
G A
GA
Buck
K M
KM
Hager
C W
CW
Naeve
R L
RL
Niece
M
M
Robertson
A J
AJ
Smith
D017931:DNA Primers; D009841:Oligonucleotides / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D009841:Oligonucleotides