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

申请资金用于购买Li-Cor 4000LS自动脱氧核糖核酸（DNA）测序仪。该设备将由不少于5个独立的项目小组使用，涉及威廉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）的教员，VIMS的研究项目小组正在进行以下研究：1)种群遗传学，生物地理学和精选海洋生物的分类学，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、少毛纲和支球纲在内的cliitellata的系统发育和系统学；4)鳟鱼主要组织相容性复合体II基因等位基因与免疫反应的关系；5)海洋无脊椎动物类群生物多样性的起源和维持。除了VIMS项目小组外，学院生物系的研究还涉及筛选基因，这些基因可以用作爪蟾的背-腹侧标记，以检查导致脊椎动物背-腹侧轴确定的组织相互作用。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。Li-Cor 4000LS自动测序仪的购买提供了相当大的优势，吞吐量和效率超过手动方法目前在威廉和玛丽学院可用。这些优点包括提高碱基识别精度，增加单个引物的读取长度，并大大减少了时间投资。自动测序仪的使用也将消除放射性废物，并将减少环境暴露的风险。目前，该学院的测序反应是在35S标记的情况下进行的。使用放射性物质会给实验人员和在同一设施工作的人带来暴露的风险，并严重减少了学生，特别是本科生获得DNA测序经验的机会。此外，废物处理也是一个问题。东弗吉尼亚现有的单一放射性废物处置场址将于1995年12月31日关闭，而一个替代设施预定要到2000年才可使用。在此期间，所有在学院产生的放射性废物必须储存在现场。在自动测序仪中红外激发的激光探测不涉及使用放射性标记方法，因此规避了由放射性废物积累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以免费使用，并且可以安全地用于任何级别的教学目的。所有排序操作的集中化将加强所有项目组之间现有的相互作用。

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.