FR AB - Science Reference

R3-M
A current profile of microarray laboratories: results of the
2000–2001 ABRF Microarray Research Group Survey of Laboratories
using microarray technologies.
G.S. Grills1, C. Griffin2, A. Massimi1, K. Lilley3, K. Knudtson4, J. VanEe5; 1Albert Einstein Col. of Med., 1300 Morris Park Avenue, Bronx,
NY 10461-1602, 2UCSF, 3Cambridge Univ., 4Univ. of Iowa,
5Cornell Univ.
The goal of this survey study was to build a current picture of the microarray
analysis world. Microarray analysis is a fast developing field. New methods
and instrumentation are being constantly introduced and the number of investigators
using the technology is rapidly expanding. We focused on Affymetrix
GeneChip and on cDNA, oligonucleotide and protein microarray
spotting technologies. Data was requested from laboratories using these
technologies by posting instructions for participation on microarray related
electronic discussion groups. A web based survey form was used to collect
information such as instrumentation, protocols, staffing, funding, and
throughput. The survey was geared toward gathering information from academic,
pharmaceutical, and commercial laboratories that offer microarray
technologies as a shared resource. Individual laboratories that have these
technologies were also invited to participate. This study is part of a continuing
survey. The data from this survey was analyzed to build a current profile
of microarray analysis laboratories.
R5-S
Preliminary results from the DNA Sequencing Research Group 2001
Study: factors that affect the sequencing and detection of mixed
base sequences in PCR products.
M.A. Robertson1, L. Hall2, J. Hawes3, T. Hunter4, E. Jackson-Machelski5, K. Knudtson6, D. Leviten7; 1Univ. of Utah, 4A 438, School of Medicine,
50 N. Medical Drive, Salt Lake City, UT 84132, 2Albert Einstein Col. of
Med., 3Indiana Univ. Sch. of Med., 4Univ. of Vermont, 5Washington Univ.
Sch. of Med., 6Univ. of Iowa, 7ICOS Corp.
With the imminent release of the draft Human Genome Sequence, many laboratories
are seeing increased demand for comparative sequencing. However,
detection of mixed bases in a DNA population is a very demanding technique
for many DNA Sequencing Core Laboratories. The DSRG will report on a
study to test the ability of the ABRF DNA sequencing community to sequence
PCR products containing several different kinds of base pair mutations, insertions
and/or deletions. To limit variables, PCR products and sequencing
primers were supplied along with a wild type reference sequence as a text
file. Members were asked to sequence the PCR products using their choice
of chemistry for mutation/mixed base detection. The set of PCR products
contained mutations of varying difficulty. The PCR products were amplified
from primers that contained universal forward and reverse primers to allow
testing of both dye terminator and dye primer sequencing chemistries. Results
were submitted as ABI chromatogram files with a short survey to gather relevant
information. Analysis of the study will address:
a) The ability of participants to be able to correctly identify the mutations.
b) The accuracy of dye primer versus dye terminator sequencing for correct
mutation detection.
c) The ability to detect mutations from only one primer sequence.
d) The differences in instrumentation in the abilty to detect mutations, e.g.,
capillary vs slab gel instruments.
e) The software/sequence alignment tools being used for mutation analysis.
RESEARCH GROUP ABSTRACTS
ABRF 2001 ABSTRACTS
R4-T
NARG 2000–2001 DNA Synthesis Study: Part I. Surveys of trends
and strategies of DNA synthesis core facilities. Part II. Evaluation of
oligonucleotide synthesis in core facilities utilizing homopolymers
of A(20), G(20), C(20), T(20) and one heteropolymer.
M.E. Gunthorpe1, J.F. Fox2, B.P. Holloway3, K.S. Lilley4, M. Lively5, K. Mills6, S.A. Scaringe7, T. Thannhauser8, A. Yeung9; 1Howard Hughes
Med. Inst., UCSF, 533 Parnassus Ave. at 3rd Ave. Rm U436, San Francisco,
CA 94143-0793, 2Univ. of Virginia, 3Ctrs. for Dis. Control and Prevent.,
4Univ. of Cambridge, 5Wake Forest Univ. Sch. of Med., 6Millennium
Pharmaceut. Inc., 7Dharmacon Res. Inc., 3200 Valmont Road #5,
Boulder, CO 80301, 8Cornell Univ., 9Fox Chase Cancer Ctr.
The Nucleic Acids Research Group (NARG) of the Association of Biomolecular
Resource Facilities (ABRF) invited subscribers to the ABRF electronic discussion
group to participate in our 2000–2001 study, which was accessed
online at the web address listed below. Part I was a general survey regarding
various aspects and issues pertaining to DNA synthesis core facilities,
such as quality control, work philosophy and the future. Part II was an evaluation
of oligonucleotide synthesis from core facilities by using a novel
approach: Isolation of synthesis problems by analysis of homopolymer
oligonucleotides. In this approach, the synthesis problems are magnified
19�–20� depending on the chemical properties of the individual bases.
Oligonulceotides submitted by participating labs will be analyzed by a range
of analytical methods including HPLC, capillary electrophoresis, MALDI-TOF
and ESI mass spectrometry to assess the quality of each product. The results
of the evaluation of oligonucleotides from participants utilizing different
instrumentation and protocols will be presented.
Web address: http://www.abrf.org/ and choose “Nucleic Acids Research
Group (NARG) 2000–2001 Study” under the category “Open Research Group
Studies”.
R6-S
Dissecting structure-function relationships in RNA/protein
interaction.
I.A. Laird-Offringa1, D.G. Myszka2, P.S. Katsamba1; 1Univ. of Southern
California, Norris Cancer Center, Room NOR 6420/1441 Eastlake Ave.,
Los Angeles, CA 90089-9176, 2Univ. of Utah, 50 N. Medical Dr./School of
Medicine Rm 4A417, Salt Lake City, Utah 84132
RNA-binding proteins play critical roles in gene expression and regulation at
the post-transcriptional level. While much is known about the various naturally
occurring RNA-binding motifs, and co-crystal structures of a number of
RNA/protein complexes are available, very little is known about the dynamics
of RNA/protein interactions. We have used the spliceosomal protein U1A
and its RNA target in the U1 small nuclear RNA (U1hairpinII or U1hpII) as a
model to study the kinetics of RNA/protein interaction. Using the previously
solved structure of the U1A/U1hpII complex, we have engineered a series of
mutants designed to probe the roles of electrostatics, hydrogen bonding, aromatic
stacking, and RNA loop length, all of which have been implicated in
formation of the U1A/U1hpII complex. The effects of these mutations on the
binding dynamics were studied using BIACORE, which yielded high quality
kinetic data about the interaction. We determined that neutralization of positive
charges on the protein slows the association rate and reduces the deleterious
effect of salt on complex formation. In contrast, removal of hydrogenbonding
or stacking interactions within the RNA/protein interface, or
reducing the size of the RNA loop, increases the dissociation rate. Our data
support a mechanism of binding consisting of a rapid initial association
based on electrostatic interactions and a subsequent locking step based on
the hydrogen bonding and stacking interactions that occur during the
induced fit of RNA and protein. Our results demonstrate the power of kinetics
to dissect the functional differences between structural features of two
interacting macromolecules.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 225