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

苏必利尔湖州立大学的埃文斯博士获得了一项拨款，用于购买一台自动基因分析仪。该设备将推进教师研究和本科生研究培训，包括DNA测序和DNA指纹识别。该项目的目标如下：1)发现各种硬骨鱼在视网膜发育过程中光感受器视蛋白基因表达的变化。2)研究在圣玛丽斯河发生的粉红鲑鱼和奇努克鲑鱼的杂交繁殖(杂交)。3)确定特定的DNA标记是否可以确定五大湖七鳃鳗是否返回它们的出生流产卵。4)为生物学和临床检验科学专业的学生提供DNA测序方面的研究培训，并对犯罪学、渔业和野生动物管理专业的学生进行DNA指纹分析技术的培训。三个教职项目将包括本科生的研究培训：1)眼睛的光谱敏感性主要由视网膜感光细胞中的视蛋白决定。在冬季比目鱼中，这些视蛋白随着动物的变态而变化。在细胞产生蛋白质之前，必须从细胞的DNA中复制信使RNA(信使RNA)。在选定的发育阶段，将检查鱼类视网膜的视蛋白mRNA表达、光感受器形态和细胞特异性视蛋白表达。利用“聚合酶链式反应”(PCR)，视蛋白基因将被扩增，然后使用遗传分析仪进行测序。一旦确定了序列，就会创建mRNA标签来跟踪鱼类视网膜从幼体到幼体状态时光感受器视蛋白的变化。2)奇努克和粉红三文鱼的杂交在圣玛丽斯河中很常见，但显然这些杂交只是奇努克雌性和粉红三文鱼雄性杂交的结果。目前尚不清楚为什么混合动力车会以这种方式发生。线粒体只从母亲那里遗传；因此，使用线粒体DNA(MtDNA)创建的DNA指纹将允许识别杂交后代的母系物种。利用遗传分析仪，将建立奇努克、粉红和“品诺”杂交鲑鱼的mtDNA指纹数据库，以确定杂交是否真的受到限制。如果是这样的话，进一步的研究将考察其中涉及的机制。3)七鳃鳗是五大湖鱼类的一种严重寄生虫。蛋白质(同工酶)分析表明，七鳃鳗回到了它们出生的产卵场所。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动的实用性。种群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。这种方法已经被用于许多鱼类种群，但不适用于七鳃鳗。遗传分析仪将被用来利用微卫星DNA标记为七鳃鳗创造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，以评估它们在监测七鳃鳗生命周期方面的有效性。4)聘请本科生担任这些项目的研究助理。此外，几门课程将包括生物学和临床检验科学专业的DNA测序，犯罪学和渔业与野生动物管理专业的DNA指纹技术。这两种方法都可供学生在他们的高中论文研究中使用。该项目对基础科学研究的几个领域做出了贡献。1)眼睛的神经视网膜是中枢神经系统的一个可接近的部分，结构和功能明显相关。了解视网膜是如何“构建”的，有助于洞察其他神经网络是如何发展的。硬骨鱼视网膜是一种典型的脊椎动物视网膜，为理解正常发育(即细胞如何分裂和分化成特定组织)提供了一个有用的模型。此外，冬季比目鱼眼睛中视蛋白基因表达的变化可能会回答细胞-细胞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，奇努克和粉红鲑鱼很少有机会杂交，但在圣玛丽斯河，‘Pinook’杂交种的数量似乎在增加。在实验室里，在两个方向上都制作了粉色和奇努克杂交，这表明杂交没有生理障碍。野生限制杂交的存在可能是领地或求偶仪式等行为障碍的结果。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，可以对基因流进行快速、常规的分析。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)在苏必利尔湖州立大学，学生通过积极参与学习是核心，教职员工认为他们的个人研究兴趣是激发学生好奇心的一种机制。DNA技术在我们的日常生活中正变得越来越突出。重要的是，我们不仅要训练学生如何在研究生涯中使用这些技术，而且要训练他们认识到这些方法何时被正确地应用和解释。

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.