DOE Human Genome Program Contractor-Grantee
7. Determining Quality of Oligonucleo-tides Synthesized in a High Throughput Process
Linda S. Thompson, David C. Bruce, Norman A. Doggett, Mark O. Mundt, and Larry L. Deaven
Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545
LANL obtained from the University of Texas Southwest Medical Center a liquid chemical dispensing robot, LCDR or Mermade, to produce large numbers of oligos. The Mermade is capable of making two 96-well plates of oligos per day. The instrument protocol adheres to the standards of DNA synthesis using deprotection, evaporation, resuspension, and quantitation. We also utilize a Biomek 2000 to resuspend the samples, prepare standard dilutions for OD readings on a plate reader, and to set up the samples for gel electrophoresis. Quality control consists of running a representative sample of the plate on BioRad 15% TBE-Urea Ready gels, 12 wells. If those samples are confirmed to be the required length without n-x species, i.e. oligos missing one or more bases, and if quantitation turns up no zero values, the plate can be given to the user. A quality control issue at this time is whether one needs to PAGE every oligo made on the Mermade. A recent random test of a plate of Mermade oligos vs. "factory made" oligos showed a success rate of 93% for both plates, indicating that 7% failed or were n-x. UTSW and LANL both report an average success rate of 95%, showing there's little or no difference between oligos made on the Mermade or those purchased through an oligo company.
LANL has also purchased a MALDI mass spectrometer. This instrument can be utilized to look at the DNA samples made by the Mermade and has been considered to take over the quality control aspect of DNA synthesis. The MALDI-MS could be used as a qualititative tool to screen all oligos for the desired length and the presence of n-x species, followed up with PAGE to identify the extent of the n-x in the suspect sequences.
|The online presentation of this publication is a special feature of the Human Genome Project Information Web site.|