An Automated DNA Sequencer for Use in Population Biology, Phylogeny, Neurobiology and Immunology

申请资金用于购买Li-Cor 4000 LS自动脱氧核糖核酸测序仪。该设备将由不少于5个独立的项目组使用，涉及威廉和玛丽学院的各种研究学科。五名主要研究人员中有四名是弗吉尼亚海洋科学研究所（VIMS）的教师，该学院的海洋科学学院位于弗吉尼亚州格洛斯特角。VIMS的研究项目组正在进行以下研究：1）选定海洋生物的种群遗传学、地理学和分类学，重点是远洋鱼类，2）单孢子虫门的生物学以及这些原生生物与其他真核生物的系统发育关系，3）包括水蛭、寡毛类和鳃蛭类在内的Clitellata的系统遗传学和系统学，4）鳟鱼主要组织相容性复合体II基因等位基因与免疫应答的关联; 5）海洋无脊椎动物类群生物多样性的起源和维持。除了VIMS项目组，在该学院的生物系的研究涉及筛选基因，可用作背腹标记爪蟾检查组织的相互作用，导致脊椎动物的背腹轴的确定。 对自动测序仪的集体需求由3个主要标准指导：1）数据吞吐量和效率，2）辐射安全性和3）集中化。购买Li-Cor 4000 LS自动测序仪与威廉和玛丽学院目前可用的手动方法相比，在通量和效率方面具有相当大的优势。这些优点包括增加的碱基识别准确性，增加的读取长度从一个单一的引物，并大大减少了时间投资。自动测序仪的使用还将消除放射性废物，并将减少环境暴露的风险。目前，在学院的测序反应是在35 S标记的存在下进行的。使用放射性材料会使实验人员和在同一设施工作的人都有受到辐射的危险，并严重限制了学生，特别是大学生获得DNA测序经验的机会。此外，废物处理也是一个问题。弗吉尼亚州东部唯一的放射性废物处置场将于1995年12月31日关闭，替代设施预计要到2000年才能建成。在此期间，学院产生的所有放射性废物必须就地储存。在自动测序仪中的红外激发的激光检测不涉及使用放射性标记方法，因此避免了由积累放射性废物和实验者暴露所施加的即将到来的限制。此外，非放射性检测方法使该技术可免费获得，并可安全用于任何级别的教学目的。所有排序业务的集中化将加强所有项目组之间的现有互动。

Funding is requested for the purchase of a Li-Cor 4000LS automated deoxyribonucleic acid (DNA) sequencer. This equipment is to be used by no fewer than 5 separate project groups involving a diverse range of research disciplines at the College of William and Mary. Four of five principal investigators are faculty at the Virginia Institute of Marine Science (VIMS), the School of Marine Science campus of the College located in Gloucester Point, VA. Research project groups at VIMS are conducting studies on 1) the population genetics, biogeography and taxonomy of selected marine organisms with emphasis on pelagic fishes, 2) the biology of the phylum Haplosporidia and the phylogenetic relationship of these protists to other eukaryotes, 3) the phylogenetics and systematics of the Clitellata including leeches, oligochaetes and branchiobdellidans, 4) the association of major histocompatibility complex II gene alleles in trout with immune responsiveness, and 5) the origin and maintenance of biodiversity in marine invertebrate taxa. In addition to the VIMS project groups, studies in the Department of Biology of the College involve screening genes that can be used as dorsal-ventral markers in Xenopus to examine the tissue interactions leading to the determination of the dorsal-ventral axis in vertebrates. The collective need for an automated sequencer has been directed by 3 principal criteria: 1) data throughput and efficiency, 2) radiation safety and 3) centralization. The purchase of the Li-Cor 4000LS automated sequencer offers considerable advantages to throughput and efficiency over manual methods currently available at the College of William and Mary. These advantages include increased base identification accuracy, increased read-length from a single primer, and greatly reduced time investment. The use of an automated sequencer will also eliminate radioactive waste and will reduce the risk of environmental exposure. Currently, sequencing reactions at the College ar e run in the presence of 35S labeling. The use of radioactive material entails the risk of exposure both to the experimenter and to those working in the same facilities and severely curtails the opportunities for students, especially undergraduates, to gain experience with DNA sequencing. In addition, waste disposal is a problem. The single radioactive waste disposal site available for eastern Virginia will close on December 31, 1995 and a substitute facility is not scheduled to be available until the year 2000. In the interim, all radioactive waste generated at the College must be stored on site. The laser-detection of infrared excitation in an automated sequencer does not involve the use of radioactive labeling methods and thus circumvents impending limitations imposed by accumulating radioactive waste and experimenter exposure. Moreover, the non-radioactive detection method makes the technology freely available and safe for teaching purposes at any level. Centralization of all sequencing operations will enhance existing interactions among all project groups.