Aquisition of a DNA Sequencer for Plant Molecular Biology Research

当前植物分子生物学研究的基础是对具有生物学和经济意义的基因进行常规测序的能力。这些基因正被克隆为来自高等植物物种的基因组和cDNA片段，以及作为植物共生体和病原体的许多微生物。分离拟南芥突变体和对特定表型改变的基因进行测序的能力，为破译植物生长和发育的分子机制提供了一种强大的新手段。不幸的是，研究生和博士后在植物分子生物学实验室的大部分实际研究时间都花在了机械而费力的程序上，通过电泳分离、检测和分析DNA测序“阶梯”。我们估计，他们至少有30%的时间被这个程序所占据，这就占用了他们更多的时间来进行更关键的、对智力要求更高的生物实验设计。本提案的目的是获得联邦配套资金购买自动DNA测序仪。这台机器将由萨斯曼教授监督，并由一位全职技术人员在他的实验室操作，薪水由威斯康辛州基金提供。这些自动化的，基于荧光的测序仪广泛应用于基因组测序项目，并已被证明是一种节省时间和劳动力的经济有效的手段。除了节省时间外，自动化技术的其他优点还包括不使用放射性核素，减少了与放射性核素安全和处置有关的经济负担。该设备的主要用户包括8名在植物分子生物学领域获得联邦资助的基础研究项目的教师。这些学院的实验室相距很近，但由5个不同的学术部门管理（农业与生命科学学院的园艺学、农学、生物化学和植物病理学部门；文学与科学学院的植物学部门）。小用户包括一些新雇佣的“应用”遗传学家，他们正在积极开展研究项目，利用重组DNA技术更有效地培育改良作物。

Underpinning much of the current research in plant molecular biology is the ability to routinely sequence genes of biological and economic interest. These genes are being cloned as genomic and cDNA fragment from higher plant species, as well as from the many microbes that act as plant symbionts and pathogens. The ability to isolate Arabidopsis mutants and sequence the genes underlying specific altered phenotypes has introduced a powerful new means of deciphering the molecular mechanisms by which plants grow and develop. Unfortunately, much of the actual research time spent by graduate students and postdoctoral associates in plant molecular biology laboratories is occupied with the rote, laborious procedure by which DNA sequencing `ladders' are electrophoretically separated, detected and analyzed. We estimate that at least 30% of their time is occupied with this procedure, which takes time away from the more critical and intellecutually demanding design of biological experiments. The purpose of this proposal is to obtain federal matching funds to purchase an automated DNA sequencer. This machine will be supervised by Prof. Sussman and operated in his laboratory by a full-time technician with salary provided by state of Wisconsin funds. These automated, fluorescent- based sequencers are widely used in genome sequencing projects, and have been shown to be a cost-effective means of saving time and labor. In addition to time savings, other advantages of the automated technique include the absence of radioactive nuclides and a decreased financial burden associated with their safety and disposal. Major users of this equipment include 8 faculty with federally funded basic research program s in the area of plant molecular biology. The laboratories of these faculty are in close physical proximity but are administered through 5 different academic departments (Departments of Horticulture, Agronomy, Biochemistry and Plant Pathology in the College of Agricultural and Life Sciences; Department of Botany in the College of Letters and Science). Minor users include several newly hired `applied' geneticists with active research programs using recombinant DNA techniques for more efficient breeding of improved crop plants.