MRI: Acquisition of Automated DNA Sequencer for Undergraduate Research and Training

MRI：采购自动 DNA 测序仪用于本科生研究和培训

• 批准号: 0116086
• 负责人: Barbara Evans
• 金额: $ 11.32万
• 依托单位: Lake Superior State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-15 至 2005-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0116086&HistoricalAwards=false
• 关键词: MRI; Acquisition; Automated; DNA; Sequencer

A grant has been awarded to Dr. Evans at Lake Superior State University to acquire an automated Genetic Analyzer. The equipment will advance faculty research and undergraduate research training involving DNA sequencing and DNA fingerprinting. The goals of the project are as follows: 1) to discover how photoreceptor opsin gene expression changes during retinal development in a variety of teleost fish species. 2) to characterize the cross-breeding (hybridization) occurring between pink salmon and chinook salmon in the St. Marys River. 3) to determine if specific DNA markers can establish whether Great Lakes sea lamprey return to their natal streams to spawn. 4) to provide research training in DNA sequencing for Biology and Clinical Laboratory Science majors, and to train students majoring in Criminalistics, and Fisheries & Wildlife Management in DNA fingerprinting techniques.Three faculty projects will incorporate undergraduate research training: 1) Spectral sensitivity in the eye is mainly determined by the opsin proteins in the photoreceptor cells of the retina. In the winter flounder, these opsin proteins change as the animals go through metamorphosis. Before a cell can produce a protein, messenger RNA (mRNA) must be copied from the cell's DNA. At selected developmental stages, fish retinas will be examined for opsin mRNA expression, photoreceptor morphology and cell specific opsin expression. Using "polymerase chain reaction" (PCR), the opsin genes will be amplified and then sequenced using the Genetic Analyzer. Once sequences are identified, mRNA tags will be created to follow the change in photoreceptor opsin as the fish retina transforms from larval to juvenile state. 2) Hybrids between Chinook and pink salmon are frequently encountered in the St Marys River, but apparently these hybrids only result from crossbreeding between Chinook females and pink salmon males. Why the hybrids are occurring this way is unclear. Mitochondria are only inherited from the mother; thus DNA fingerprints created using mitochondrial DNA (mtDNA) will allow the maternal species of the hybrids to be identified. Using the Genetic Analyzer, a database of mtDNA fingerprints will be established for the chinook, pink and "pinook" salmon hybrids to determine if the hybrid crosses are truly restricted. If so, further studies will examine the mechanisms involved. 3) Sea lamprey are a serious parasite on native Great Lakes fishes. Protein (isozyme) analysis has indicated that lamprey return to their natal spawning grounds. The current study will test the utility of using DNA fingerprints of lamprey populations to assess the gene flow between populations. Population genetic surveys commonly create DNA fingerprints using microsatellite DNA, which are unique regions of the DNA. Such methods have been used for many fish populations, but not for lamprey. The Genetic Analyzer will be used create DNA fingerprints for the lamprey using microsatellite DNA markers. The results from these genetic analyses will be compared with the previous isozyme studies to assess their usefulness for monitoring lamprey life cycles. 4) Undergraduate students will be hired as research assistants on these projects. In addition, several courses will incorporate DNA sequencing for Biology and Clinical Laboratory Science majors and DNA fingerprinting technology for Criminalistics, and Fisheries & Wildlife Management majors. Both techniques will be available for students to use in their senior thesis research. The project contributes to several fields of basic science research. 1) The neural retina of the eye is an accessible part of the central nervous system where structure and function are clearly related. Understanding how the retina is "built" gives insight into how other neural networks develop. The teleost fish retina is a typical vertebrate retina and presents a useful model for understanding normal development (i.e., how cells divide and differentiate into specific tissues). In addition, the change in opsin gene expression in the winter flounder eye may answer questions of how cell-cell interactions determine a cell's fate. 2) In their natural habitat, there are few opportunities for Chinook and pink salmon to crossbreed, but in the St. Marys River, the number of 'pinook' hybrids appear to be increasing. In the laboratory, pink and chinook crosses have been made in both directions suggesting no physiological barriers to hybridization. The presence of restricted crossbreeding in the wild may be a result of behavioral barriers such as territoriality or courtship rituals. 3) Sea lampreys are a major problem in the Great Lakes, and a solution may reside in understanding their life history and spawning behavior. Protein analysis indicates that lamprey do return to the region of their birth to spawn. Use of microsatellite DNA markers to create DNA fingerprints is a method that can be automated, allowing fast, routine analysis of gene flow. A better understanding of lamprey population genetics may allow these populations to be brought under control in the future. 4) Student learning, by active participation, is central at Lake Superior State University, and faculty consider their personal research interests to be a mechanism to stimulate student curiosity. DNA technology is becoming increasingly prominent in our everyday lives. It is important that we not only train students how to perform these techniques for research careers, but also train them to recognize when these methods are being applied and interpreted correctly.

已向苏必利尔湖上级州立大学的埃文斯博士提供赠款，以购买一台自动遗传分析仪。该设备将推进涉及DNA测序和DNA指纹识别的教师研究和本科生研究培训。本课题的主要目的是：1）研究不同硬骨鱼类视网膜发育过程中感光细胞视蛋白基因表达的变化。2)以表征在圣玛丽河中发生的粉红鲑鱼和奇努克鲑鱼之间的杂交（杂交）。3)以确定特定的DNA标记是否可以确定五大湖海七鳃鳗是否回到它们的纳塔尔河流产卵。4)为生物学及临床实验科学专业的学生提供DNA测序的研究训练，并为犯罪侦查学及渔业及野生动物管理学专业的学生提供DNA指纹技术的训练。三个学院项目将纳入本科生的研究训练：1）眼睛的光谱敏感度主要由视网膜感光细胞中的视蛋白决定。 在冬季比目鱼中，这些视蛋白质随着动物的变态而变化。在细胞可以产生蛋白质之前，信使RNA（mRNA）必须从细胞的DNA复制。在选定的发育阶段，将检查鱼视网膜的视蛋白mRNA表达、光感受器形态和细胞特异性视蛋白表达。使用“聚合酶链反应”（PCR），视蛋白基因将被扩增，然后使用遗传分析仪进行测序。一旦序列被确定，mRNA标签将被创建以跟随感光体视蛋白的变化，因为鱼视网膜从幼虫状态转变为幼年状态。2)奇努克和粉红鲑鱼之间的杂交经常在圣玛丽河遇到，但显然这些杂交只会导致奇努克女性和粉红鲑鱼男性之间的杂交。为什么混血儿会以这种方式出现还不清楚。线粒体只从母亲遗传;因此，使用线粒体DNA（mtDNA）创建的DNA指纹将允许杂交种的母系物种被识别。使用遗传分析仪，将建立一个数据库的线粒体DNA指纹的奇努克，粉红色和“皮努克”鲑鱼杂交，以确定是否真正限制杂交。如果是这样的话，进一步的研究将检查所涉及的机制。3)海七鳃鳗是一种严重的寄生虫对本地五大湖鱼类。蛋白质（同工酶）分析表明，七鳃鳗返回其纳塔尔产卵场。目前的研究将测试使用七鳃鳗种群的DNA指纹来评估种群之间的基因流的效用。群体遗传调查通常使用微卫星DNA创建DNA指纹，微卫星DNA是DNA的独特区域。这种方法已被用于许多鱼类种群，但不是七鳃鳗。遗传分析仪将使用微卫星DNA标记为七鳃鳗创建DNA指纹。这些遗传分析的结果将与以前的同工酶研究进行比较，以评估其监测七鳃鳗生命周期的有用性。4)本科生将被聘为这些项目的研究助理。此外，几门课程将纳入生物学和临床实验室科学专业的DNA测序，以及犯罪学和渔业野生动物管理专业的DNA指纹技术。这两种技术将可供学生在他们的高级论文研究中使用。该项目有助于基础科学研究的几个领域。1)眼睛的神经视网膜是中枢神经系统的可接近部分，其中结构和功能明确相关。了解视网膜是如何“构建”的，可以深入了解其他神经网络是如何发展的。硬骨鱼视网膜是典型的脊椎动物视网膜，并且提供了用于理解正常发育的有用模型（即，细胞如何分裂和分化成特定的组织）。此外，冬比目鱼眼睛中视蛋白基因表达的变化可能会回答细胞间相互作用如何决定细胞命运的问题。2)在它们的自然栖息地，奇努克和粉红鲑鱼杂交的机会很少，但在圣玛丽河，“皮努克”杂交鱼的数量似乎正在增加。在实验室中，粉红色和奇努克杂交已经在两个方向上进行，表明杂交没有生理障碍。在野外限制杂交的存在可能是行为障碍的结果，如领土或求偶仪式。3)七鳃鳗是五大湖的一个主要问题，解决办法可能在于了解它们的生活史和产卵行为。蛋白质分析表明七鳃鳗确实会回到它们出生的地方产卵。使用微卫星DNA标记来创建DNA指纹是一种可以自动化的方法，可以快速，常规地分析基因流。对七鳃鳗种群遗传学的更好理解可能会使这些种群在未来得到控制。4)学生的学习，通过积极的参与，是中央在苏必利尔湖上级州立大学，教师认为他们的个人研究兴趣是一种机制，以激发学生的好奇心。DNA技术在我们的日常生活中变得越来越重要。重要的是，我们不仅要训练学生如何为研究事业执行这些技术，而且还要训练他们认识到这些方法何时被正确应用和解释。