FR AB - Science Reference

ABRF 2001 ABSTRACTS
P17-M
New methods for the synthesis of multiple oligonucleotide
sequences either singly or linked end-to-end in tandem.
R.T. Pon1, S. Yu1, Y.S. Sanghvi2; 1Univ. of Calgary, 3350 Hospital Dr. NW,
Calgary, AB T2N 4N1 Canada, 2Isis Pharmaceut.
Solid-phase oligonucleotide synthesis requires attachment of the 3�-terminal
nucleoside to the support, usually through a 3�-ester linkage. Phosphoramidite
methods for performing this attachment through a phosphate linkage
on “Universal” supports have been developed. However, these methods
introduce a terminal 3�-phosphate residue which is difficult to remove. We
have developed new linker phosphoramidite reagents which eliminate this
problem by incorporating a cleavable 3�-ester linkage within the linker arm.
Thus, only products with 3�-OH ends are produced and no extra conditions
for 3�-dephosphorylation are required. These new reagents provide an alternative
to our previous method for automated nucleoside attachment using
HBTU and DMAP (Pon and Yu, Synlett 1999, 1778). The linker phosphoramidites
are especially suited to high-throughput synthesis in 96-well plates
since inexpensive, underivatized CPG can be used as a “Universal” support.
Nucleoside attachment to the 5�-OH group of a prior sequence is also possible.
This allows multiple oligonucleotides linked end-to end in tandem to
be prepared in a single synthesis. The linked oligonucleotides are released
from each other upon cleavage from the support and used together as a mixture.
Sequences prepared in tandem can be PCR primers, forward and
reverse sequencing primers, or duplex DNA fragments. The total number of
bases is only limited by the support’s pore size. We have prepared strings of
oligonucleotides up to 96 bases long (4 � 24 base primers) and combinations
of up to ten shorter sequences.
P19-S
Production of antigen specific MHC class I tetramers as a core
facility service.
P.S. Adams, T.B. Miller, M.J. Dobrzanski, R.J. Hogan, D.L. Woodland;
Trudeau Inst., 100 Algonquin Avenue, Saranac Lake, NY 12983
Major Histocompatibility Complex (MHC) Class I tetramers are composed of
a tetrameric complex of the MHC Class I molecule and a specific antigenic
peptide. The Class I heavy chain has been modified to contain a BirA
sequence that allows biotinylation and subsequent tetramerization of the
complex with a fluorescently tagged strepavidin. These tetramers can be used
to identify antigen specific CD8� T cells involved in immune responses using
flow cytometry. The Molecular Biology Core Facility prepares these tetramers
as a core facility service to aid the researchers at the Trudeau Institute. One
project involves the characterization of the underlying mechanisms and
immunoregulatory roles of tumor antigen-reactive Tc1 and Tc2 effector cell
sub-populations, which can provide insight into the modification and/or
development of novel immunotherapeutic approaches to disseminated malignancies.
Another project involving viral immunity has demonstrated that a
substantial population of antigen specific CD8� T cells can be found not
only in the secondary lymphoid organs, but also in the peripheral tissues after
resolution of a primary respiratory virus infection. These data have significant
implications for vaccines that induce systemic rather than local immunity. The
production procedure and examples of the usefulness of MHC Class I
tetramer technology will be presented.
POSTER ABSTRACTS
192 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P18-T
Performance of high-speed oligonucleotide synthesizer in 96-well
format.
D. Luk, A. Tuyet-Doan, S. Hall, J. Koh, R. Guettler; GeneMachines,
935 Washington St., San Carlos, CA 94070
Oligonucleotides are essential elements of genomic research. The Poly-
PlexTM oligonucleotide synthesizer makes 96 different oligos in standard 96well
format, which is amenable to downstream high-throughput processing
and handling. Operator time is minimal and synthesis time is less than 3
hours for a 96-well plate of 20-mers. By eliminating flushing of reagent lines
through a parallel dispensing technology, optimal synthesis time and reagent
consumption are achieved. PolyPlex’s low reagent consumption generates
oligos quickly, at low costs and small scales as low as 10 nmol. Synthesis
costs, including all consumables, are less than $0.10 per base. The PolyPlex
synthesis chamber provides an inert-gas environment where synthesis
progress can be monitored by using full-plate trityl collection after any base
addition. PolyPlex utilizes fully licensed chemistry and generates oligos with
greater than 98% coupling efficiency. Versatile, easy-to-use software provides
powerful synthesis control along with extreme simplicity.
P20-M
Advantages of directionally immobilized 6xHis-tagged proteins
for capture assays.
K. Steinert, F. Schäfer, S. Wahle, A. Hoffer, P. Söhnlein, J. Ribbe; QIAGEN
GmbH, Max-Volmer-Strasse 4, Hilden, Nordrhein-Westfalen 40724,
Germany
Assay signals and reproducibility can be greatly enhanced by immobilizing
the assay components in a directed manner. For example, to set up an interaction
assay it is essential that the immobilization of the capture molecule
occurs in active conformation to allow binding of the interaction partner. Similarly,
oriented binding in immunoassays results in optimal accessibility of
antigenic regions and therefore increased signal strengths. Directed immobilization
of assay components can easily be achieved by using 6xHis-tagged
recombinant proteins and immobilizing them via their 6xHis tags. The use of
this interaction is well established for efficient purification of 6xHis-tagged
proteins, but is also advantageous for immobilization of recombinant proteins
in highly sensitive and reproducible protein-based assays. In addition, relying
on the 6xHis-Ni-NTA interaction for immobilization of assay components
simplifies assay development, because immobilization conditions no
longer have to be optimized for each individual protein, but rely on the same
biochemical characteristics of the 6xHis-Ni-NTA interaction for each protein.
As an alternative to Ni-NTA, monoclonal antibodies recognizing the 6xHis tag
can be used for highly specific and oriented immobilization of 6xHis-tagged
proteins. The effect of specifically oriented binding on assay results is demonstrated
by comparison of ELISA experiments employing proteins passively
adsorbed to polystyrene plates and proteins immobilized via their 6xHis tags,
as well as by a study on the interaction of two chaperones and development
of a protease assay.