FR AB - Science Reference

P13-S
Microsatellite analysis using fluorescent PCR primers synthesized
in tandem.
S.L. Wegener, G.J. Wiebe, S. Yu, R.T. Pon; Univ. of Calgary,
3330 Hospital Drive N.W., Calgary, AB T2N 4N1, Canada
A new procedure for solid-phase synthesis of multiple oligonucleotides
linked end-to-end (tandem synthesis) on the same solid-phase support has
recently been developed in our laboratory. Upon cleavage and deprotection,
the oligonucleotides are released from each other yielding a mixture of two
or more sequences in the same solution. This procedure has practical benefits
for applications requiring large sets of oligonucleotides, especially PCR
amplification, since the number of individual oligonucleotide syntheses to be
set-up and the number of individual oligonucleotide samples handled is
reduced by half. Fluorescently labelled primer pairs for automated genotyping
are also possible by incorporating a fluorescent dye phosphoramidite
onto the 5�-end of the terminal oligonucleotide. PCR primer pairs with FAM,
HEX, and TET fluorescent labels have been prepared by tandem synthesis
and used to amplify known microsatellite markers from (C57BL/6 � NOD)
mouse genomic DNA. Automated microsatellite analysis using GeneScan
and an Applied Biosystems Prisim 377 DNA sequencer was then performed.
P15-T
Quality control in a core oligonucleotide synthesis facility.
R.R. Muhlhauser, C.G. Miller, A. Yeung; Fox Chase Cancer Ctr.,
7701 Burholme Avenue, Philadelphia, PA 19111
Our facility makes about 400 oligonucleotides each month. For the past 16
years, we have done quality control on each oligonucleotide prior to delivery.
Our users do not want the quality of oligonucleotides to ever be a variable
for troubleshooting in their experiments. Even a failure rate of 1% in
oligonucleotide synthesis would have caused four experiments to fail each
month. Such a low rate of synthesis failure is difficult to isolate in a statistical
approach of quality control by random sampling. Analyzing every
oligonucleotide also allows us to catch instrument and reagent problems
more quickly. By analyzing each oligonucleotide immediately after deprotection,
we reject 1 to 3% of the oligonucleotides we make that do not meet
our quality standard. These oligonucleotides are resynthesized without delay.
The oligonucleotide analysis method we use is anion-exchange at pH 12.5,
using the Mono Q system on an FPLC (Yeung, A. T. and Miller, C. G. Anal.
Biochem. 187:6675, 1990), assisted by a home-made autosampler and autoinjector.
At that pH, both n-1’s and deprotection problems are visible. We
highly recommend this practice because it does not add appreciable expense
or effort to the cost of the oligonucleotide, but provides the facility personnel
with assurance in the quality of their products.
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P14-M
Economical usage of value-added phosphoramidites.
J.B. Hobbs; Univ. of British Columbia, #237-6174 University Boulevard,
Vancouver, BC V6T 1Z3, Canada
A method is described for economical use of value-added (dye, biotin, etc.)
phosphoramidites. The method was developed on an Applied Biosystems
380B synthesizer. The amidite is placed in a glass autosampler vial which is
located in a plastic jacket inside a 10 ml glass vial. The amidite is dissolved
in situ using a user-variable microdilution adapted from a bottle-change procedure.
Typically 5 mg. of amidite are dissolved in 100 microlitres of acetonitrile.
The amidite is used in conjunction with Applied Biosystems LV
columns and synthesis cycles adapted for use with LV columns which use a
similar “multiple-coupling” approach to that seen in “LV cycles” on later (392,
394, etc.) Applied Biosystems synthesizer models. These cycles are also
described. Good tagging efficiency can be obtained for 40 nmole or 200
nmole scales using 5 mg. of amidite in this way, and the wastage of large
quantities of expensive amidites is avoided. While the methods are applicable
to later model synthesizers, the relatively modest flow rates, gas pressures
and line volumes of the 380B render it a good platform for processes of this
type.
P16-S
Incorporation of pseudouridine and 4-thio-uridine into RNA
oligonucleotides using 5�silyl-2�-ACE-orthoester chemistry.
S.A. Scaringe1, D. Kitchen2, J. Qui2; 1Dharmacon Res. Inc.,
3200 Valmont Road #5, Boulder, CO 80301, 2Dharmacon Res., Inc.,
Boulder
The ability to incorporate a wide range of modified ribonucleotides into RNA
oligos is an essential requirement for RNA chemical synthesis methodologies.
Many modified bases have already been incorporated into RNA oligos using
5�-Silyl-2�-ACE-orthoester chemistry. We report here the incorporation of
two more important modified bases, pseudouridine and 4-thio-uridine.
Pseudouridine was converted from the nucleoside to the protected nucleoside
phosphoramidite in five steps in 45% overall yield. During synthesis the
amidite coupled in 99% stepwise yields. The high yields for both the amidite
synthesis and the oligonucleotide coupling have allowed pseudouridine to
be incorporated into RNA on a routine basis. For example, a series of RNA
oligonucleotides containing 1–3 pseudouridines were synthesized to study
the 1920 loop region of E. coli 23S RNA.
4-Thio-uridine was incorporated into RNA oligos using the convertible nucleoside
approach. The protected 4-triazole-uridine phosphoramidite was synthesized
from uridine in six steps in 32% yield. The amidite coupled in 99%
yields. Following oligo synthesis the support bound oligonucleotide was
treated with thioacetic acid, buffered to pH 8.0, for 6 hours at 55�C. The support
was washed and then treated with 10% DBU methanol at room temperature
for 24 hours. The oligonucleotide was desalted and 2�hydroxyl
protecting groups were removed using a pH 3.8 aqueous buffer for 30 minutes
at 55�C. The resulting oligonucleotides contained 98% 4-thio-uridine at
the desired position as assayed by anion exchange HPLC analysis. This is significantly
higher than the results using cyanoethyl protection of 4-thiouridine.
All oligonucleotides containing 4-thio-uridine clearly exhibited the
characteristic 330 nm absorption peak. 4-Thio-uridine is now being routinely
incorporated in RNA oligonucleotides for use in several collaborative
studies.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 191