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Increasing Oligonucleotide Analysis Throughput: Serial Injection before Analysis on the Beckman P/ACE MDQ
Oligonucleotides were synthesized with the Bioautomation MerMade multiplex oligonucleotide synthesizer, using standard phosphoramidite chemistry. After deprotection, dehydration and Sephadex G50 cleanup, the oligonucleotides were suspended in 100 l of deionized ultrafiltered (DIUF) H20, and the concentration of each determined by UV spectroscopy using a SpectraMax UV-Vis 96-well spectrophotometer. An aliquot of each oligo was then diluted to approximately 2pmol/ l in DIUF H20, in a Beckman-Coulter 96-well microtiter tray, and covered with 2-3 drops of mineral oil.
Preparation of CE materials For oligonucleotide separations, we used the Beckman eCAP ssDNA kit, which included capillaries (eCAP DNA 100 m internal diameter), buffer (TRIS/borate), urea, lyophilized sieving gel matrix, and Test mix pd (A) 40-60 (an oligonucleotide standard). The capillary was prepared according to the manufacturers instructions, to make a final capillary of 30 cm total length and 20 cm from the inlet to the detector. The buffer components were hydrated with deionized water (Milli-Q) to make 130 ml of the TRIS/borate buffer, according to the instructions. Solid urea pellets were added slowly to the buffer, with stirring, to a final concentration of 7 M. Five ml of this urea/buffer solution was then added to the lyophilized gel matrix, and stirred for 5 hours to dissolve. Test mix pd(A) 40-60 was suspended in 500 l of sterile DIUF water and placed in 100 l aliquots in Beckman-Coulter micro vials. Before loading on the machine, vials of buffer and water were degassed in a vacuum oven at 20kilopascals negative pressure and room temperature for 10 min. In addition, sieving gel and test mix micro vials were centrifuged for 2 min at 15,000 x g.
The SIBA protocol The Beckman Coulter P/ACE MDQ capillary electrophoresis instrument was equipped with a peltier-thermostatted sample chamber and ultraviolet (UV) detector with filtered, single wavelength detection capability, performed at 254nm for oligo detection. The sieving gel matrix was placed in a Beckman Coulter micro vial, and then placed in position A1 of the Sample Outlet (SO) tray, and the oligonucleotide sample tray was placed in the Sample Inlet (SI) position. The A1 position of the Buffer Inlet (BI) tray and Buffer Outlet (BO) tray contained vials with 1.6 ml of DIUF water. Waste vials containing 800 µl of DIUF water were placed at BI tray F6 and BO tray B1 to catch waste purged from the capillary during the capillary rinsing and equilibration steps. BI tray and BO tray A2 through A6 contained 1.5 ml vials of the TRIS/borate/urea separation buffer. 100 l of Test mix pd (A) 40-60 was placed in a Beckman-Coulter micro vial and positioned in BI F1. The remaining positions of the BI and BO trays were empty. To start the electrophoresis run, the capillary was briefly rinsed with DIUF water, from BI position A1 to BO position B1 (waste), at 20psi for 1 minute followed by rinsing with separation buffer, from BI position A2 to BO position B1, at 20psi for 1 minute. The sieving gel was then introduced into the capillary by applying a reverse pressure rinse from position SO A1 to position BI F6 (waste) at 50psi, for 10 minutes. The voltage was applied at 3 kV for 5 minutes, with separation buffer at the inlet and outlet of the capillary (BI A2, BO A2). Then, a 10-minute equilibration at 9 kV was applied using the same inlet and outlet separation buffer. After the equilibration step, the oligonucleotide standard was electrokinetically injected into the capillary at 10kV for 2 sec. Pressure was applied at 20psi during the separation at 9kV for 45 min, followed by a water dip of the capillary inlet and outlet. During electrophoresis, the capillary temperature was kept constant at 30oC, while the sample compartment was thermostat-controlled to 8oC for the remainder of the analysis. The temperature was kept low to preserve the integrity of the sieving gel, which will break down with time, resulting in a loss of resolution. The first oligonucleotide was introduced into the capillary by electrokinetic injection at 10 kV for 2 seconds, followed by a water dip of the capillary inlet and outlet. Pressure was applied at 20psi to both the inlet and outlet of the capillary during the separation at 9 kV to minimize loss of the capillary sieving gel to the buffer vials and also to minimize outgassing of the gel. The oligonucleotide was electrophoresed into the capillary at 9kV for 5 minutes, whereupon the capillary was subjected to a second water dip. A second oligonucleotide was introduced into the capillary by a second cycle of electrokinetic injection and water dip to eliminate carryover. This cycle of electrophoresis, electrokinetic injection and water dip of the capillary was then repeated an additional three times; a total of five oligonucleotides were introduced into the capillary prior to the movement of the first oligo past the detector. This cyclic injection-electrophoresis-injection procedure was performed four times, allowing the injection and analysis of 20 oligonucleotide samples before fresh gel matrix was introduced into the capillary and equilibrated. To change gel matrices, reverse pressure was applied to SO A1, containing the sieving gel, for 10 minutes at 50psi, removing the used gel from the capillary to the waste vial at position BI F6. Following gel replacement, the new gel was equilibrated for 10 minutes at 9 kV. This "gel replacement" procedure was programmed as an individual method in the P/ACE MDQ Software. The next 20 oligonucleotides were electrophoresed using the next set of separation buffers (i.e., BI A3 and BO A3 would be the next separation buffers following BI A2 and BO A2 separation buffers).
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As seen in ...
Figure A
Capillary electrophoresis of oligonucleotides using the SIBA protocol. Figure A shows the electropherogram of five high-quality 25-mer oligonucleotides. Based on peak area analysis, the full-length oligonucleotide products were present at approximately 65% of the total (mean 65.4%, std. dev. 3.4%; range of 60.4-70.9%). This is the expected abundance of the full-length product for oligonucleotide synthesis at >98-99% coupling efficiency. These oligonucleotides were subsequently shown to be successful in PCR amplification.
Figure B
In contrast to Figure A, this figure shows the results of an electropherogram of low quality 25-mers; full-length products were only a mean of 16.4% of the total products seen on the electropherogram (std. dev. 11.8%; range of 0.1-21.5%). These PCR primers subsequently failed to successfully amplify in a standard PCR reaction. These data were taken as screen captures of the electropherograms presented by the Beckman MDQ software. Both color images were imported into Graphic Converter v 4.0 on a Macintosh dual G4 computer, trimmed, and text added.
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