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ABRF 2001 ABSTRACTS POSTER AND RESEARCH GROUP ABSTRACTS P145-S Effects of irradiation on the structure of proteins. K. Balamurugan, D-H. Chin; Natl. Changhua Univ. of Educ., Post Doctor, c/o Prof. Der-Hang Chin, Department of Chemistry, National Changhua University of Education, Changhua 50058, Taiwan In this study, we investigate the effects of irradiation on protein samples in optical spectrometers. The protein samples exposed to light in a spectrofluorometer are found to undergo in situ photoreactions. Continuous irradiation of proteins resulted in progressive decrease in the emission intensity. The light-induced changes are irreversible and appear to be oxygen-dependent. Continuous exposure to light renders the protein susceptible to cleavage. The secondary structural contents in proteins show appreciable decrease upon irradiation. In addition, the temperature-induced unfolding of proteins appear to be significantly influenced by the photoreactions caused due to irradiation. R1-M Fragment Analysis Research Group Study 2001: enhancing automated allele calling by tailing dinucleotide repeat markers to eliminate nontemplate driven nucleotide addition seen with Taq polymerase. D.A. Bintzler1, P.S. Adams2, L.W. Ballard3, Y. Bao4, D. Bartley5, L. Kasch6, L.O. Petukhova7, C. Rosato8, C.E. Terrell1; 1Univ. of Cincinnati, 231 Bethesda Avenue, Cincinnati, OH 45267-0524, 2Trudeau Inst., 100 Algonquin Avenue, Saranac Lake, NY 12983, 3Univ. of Utah, 4Univ. of Virginia, 5Johns Hopkins Univ., 6John Hopkins Univ., 7Rockefeller Univ., 8Oregon State Univ. Fragment analysis is a growing field in the genetic sciences. Some basics in methodology may provide useful tools to the many service facilities expanding to include fragment analysis. This tutorial will be an instruction based discussion covering basic methodology and challenges associated with fragment analysis using slab gel and capillary electrophoresis. Topics will include methods for producing clean PCR products through PCR optimization for single markers and multiplexing markers before and after PCR. Instrument related factors that may affect the quality of the data would be discussed for both slab gel and capillary electrophoresis platforms. Finally, a section covering analysis of the results will discuss improving accuracy through selecting correct size standard peaks and developing the best standard curve. Methods of calculation will be compared. Means to properly identify the correct fragment peak from the multiple peaks commonly seen during the fragment analysis run will be demonstrated. Participants in the tutorial will come away with methods designed to improve their capabilities to start, or continue Fragment Analysis in their laboratory. 224 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 P146-M Improving productivity in core facilities using the BioRobot 3000. F. Siegman, H. Kijenski, C. Schade, A. Wehren; QIAGEN Inc., 28159 Avenue Stanford, Valencia, CA 91355 Core facilities providing molecular biology services often require automated systems to perform routine tasks. To meet the various demands faced by a core facility, these workstations should provide the flexibility to automate a wide variety of applications as well as the ability to exchange data with other laboratory instruments and data management systems. The BioRobot 3000 is a series of custom-designed workstations for automating routine tasks such as nucleic acid purification, protein purification and assay, and other liquidhandling tasks including reaction setup and sample rearray. Each workstation is tailor-made to meet individual application requirements. Different BioRobot 3000 configurations designed for different applications will be described. A high-throughput sample purification system with 4 integrated vacuum manifolds provides rapid processing of 384 samples in a single run. Up to 6 �g DNA (plasmids, cosmids, BACs, PACs, or P1 constructs) are purified per well in about 1 hour and 15 minutes using optimized protocols which minimize operator interaction and runtimes. Areas outside the BioRobot 3000 worktable can be accessed using configurations with an extended arm. This arm allows the robotic handling system to move labware to external instruments, such as spectrophotometers and thermal cyclers. The operating system uses standard data formats to facilitate data exchange with these instruments as well as data management systems, allowing samples to be tracked as they move through various processing and analysis steps. R2-M PSRG 2001 Study: synthesis of difficult sequences. N.P. Ambulos, Jr. 1, L. Bonewald2, S. Kates3, G. Osapay4, H. Remmer5, A. Somogyi6, G. Tsaprailis6, S. Vigil-Cruz7; 1Univ. of Maryland Sch. of Med., 655 W. Baltimore St. BRB13-009, Baltimore, MD 21201, 2Univ. of Texas Hlth. Sci. Ctr., San Antonio, 3Consensus Pharmaceut., Inc., 4Univ. of California, Irvine, 5Univ. of Illinois, 6Univ. of Arizona, 7Univ. of Connecticut Particular peptide sequences are inherently a challenge to solid-phase synthetic methodologies as a result of concomitant side-reactions and/or conformational issues that may result in the failure to generate the desired product. Development of improved synthetic methodologies, reagents and amino acid derivatives are offering new solutions to the old problem of difficult sequences. This year’s ABRF Peptide Synthesis Research Group designed a study to evaluate how participating laboratories solve the challenge of correctly synthesizing these troublesome peptides. The laboratories were asked to prepare the following peptide that has several potential synthetic obstacles. H-Gln-Thr-Ser-Ser-Gly-Thr-Thr-Ser-Trp-Val-Thr-Ser-Arg-Arg-Asp- Gly-Ala-Gly-Ala-Gly-Pro-OH The peptide was synthesized and both crude and purified material was analyzed and characterized by the members of the Peptide Synthesis Research Group by HPLC, AAA, MALDI-MS and ESI-MS. Both crude and purified samples were requested from participating laboratories and were also characterized by HPLC, AAA, MALDI-MS and ESI-MS. Analysis of the samples also included a comparison of yield of correct product during purification. The results of these analyses will be presented.
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
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RESEARCH GROUP REPORTS Strategies f
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TABLE 1 Summary of Results from ABR
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REFERENCES 1. Angeletti RH, Bonewal
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