High Throughput Microrepository for Genetic Materials

描述（申请人提供）：认识到迫切需要一种安全、快速和可靠的手段来保存和归档遗传样本和其他生物材料，如合成DNA样本、独特的分离样本、质粒、法医样本、患者样本、确保动植物未来生物多样性的样本、克隆和考古样本，NIH NCRR最近召集的一个研究小组确定了最迫切需要的领域，并提出了几项建议。他们的主要建议清单上的第一和第五项是“鼓励开发高通量和可扩展的种质加工和冷冻保存技术”和“支持将开辟新天地的新型'高风险/高回报'保存技术”。我们已经直接和成功地解决了这些需求与我们的微库遗传材料（MGM）技术，提供完全可扩展的，非常高密度的样品存储与自动检索从阵列的光学多路复用编码珠通过提供样品澄清，识别，存档，检索功能在一个单一的平台。在第一阶段的工作中，一个完全自动化的原型MGM平台成功地建立和测试。MGM包含多孔玻璃或聚合物珠的密集阵列，较小的珠或纸样本的组，其中每个包含不同的DNA样品，其已经用我们的基于稀土的Parallume技术进行光学编码，该技术允许每个珠被唯一地光学识别。在将DNA放置到任一单个大珠上之后，将珠加载到珠定位载玻片（BLS）中，其将珠光学隔离成平面单层用于成像，并且读取每个珠的光学代码以确定该特定DNA样品的位置。取出微珠并置于所需位置，通过qPCR确认处理期间无污染。通过进行额外的实验，包括采用新的改进硬件设计，并成功改变Parallume编码材料的化学成分，以防止紫外线对DNA的损伤，我们已经解决了所有评审员的担忧。我们现在已经构建并成功测试了一个功能完整的原型MGM仪器，并且，为了响应有洞察力的评审员的评论，我们已经修改了Parallume材料，以便允许用更长波长的激发光（365 nm）进行激发，这（a）防止在光学解码样品期间DNA的光化学分解，（B）允许使用非UV光学器件来聚焦光，以及（c）允许使用更亮、寿命更长且更便宜的LED光源。一种新的表面化学，它强烈地将DNA结合到珠上，直到它被化学释放，将提供另一个水平的样品污染保护。这里描述的改进的MGM代表了自该领域开始以来在解决DNA和核酸的快速扩展的存储需求方面的第一个实质性进展。采用光学编码珠技术的新存储范例提供了当前不可能的样品密度、自动化程度和简单性，并且具有可扩展性，以在可预见的未来成功地存储和检索任何数量的DNA样品。 公共卫生相关性：近年来，从生物体中合成或分离的遗传物质（如DNA）样本数量大幅增加。其中一些样本，如个人的DNA，法医样本或来自新细菌菌株的遗传物质是独一无二的，无法替代。然而，目前还没有以自动和高通量的方式组织或自动存档和检索DNA样品的手段。由于实验室、政府、医院和大学的吞吐量增加，预计这个问题在未来只会越来越严重。 我们在这里提供了一个新的仪器平台的设计，指定为遗传物质的微库（MGM）。样本将储存在多孔玻璃珠阵列中，每个玻璃珠显示光学代码或签名，可唯一识别特定样本和该玻璃珠内的DNA。将DNA样品各自置于直径为50微米（0.05mm）的单个高度多孔珠粒中。微珠采用我们的ParallumeTM技术进行光学编码，可通过微珠激发时发出的不同颜色的强度比来识别每个微珠。因此，成千上万的珠子可以混合在一起，每个珠子都由其独特的光学代码识别，并使用光纤将其从存储阵列中取出。在通过检查当珠被光纤拾取时发射的光来检查光学特征之后，珠可以被放置在期望的目的地位置。因此，我们能够设计和建造一个储存库，可以以一种稳健和廉价的方式存储数十万个DNA样本。这种MGM技术是完全可扩展的，在可预见的未来应该提供快速准确的DNA存储和检索能力。

DESCRIPTION (provided by applicant): High Throughput Microrepository for Genetic Materials Recognizing the urgent need for a safe, rapid and reliable means of preserving and archiving genetic samples and other biological materials, such as synthetic DNA samples, unique isolated samples, plasmids, forensic samples, patient samples, samples to ensure future biodiversity in plants or animals, clones and archeological samples, a study panel recently convened by NIH NCRR identified the areas of most critical need and made several recommendations. The 1st and 5th items on their list of major recommendations were to "encourage the development of high throughput and scalable technologies for Germplasm processing and cryopreservation" and "support novel 'high risk/high return' preservation technologies that will break new ground". We have directly and successfully addressed these needs with our Microrepository for Genetic Materials (MGM) technology which affords completely scalable, very high density sample storage with automated retrieval from arrays of optically multiplexed encoded beads by providing sample preparation-identification-archiving-retrieval functions on a single platform. For the Phase I effort, a completely automated prototype MGM platform was successfully built and tested. The MGM contains dense arrays of porous glass or polymer beads, groups of smaller beads or paper swatches, each of which contains a different DNA sample, which have been optically encoded with our rare earth-based Parallume technology which allows each bead to be optically identified uniquely. After placement of the DNA onto either single large beads, the beads are loaded into Bead Localization Slides (BLS), which optically isolate the beads into a planar monolayer for imaging, and the optical code of each bead is read to determine the location of that particular DNA sample. The beads are retrieved and placed in the desired location and the lack of contamination during handling confirmed by qPCR. By performing additional experiments, including incorporation of new improved hardware designs and successfully changing the chemical composition of the Parallume encoding materials to prevent UV damage to the DNA, we have addressed all Reviewers' concerns. We have now built and successfully tested a completely functional prototype MGM instrument and, in response to the insightful Reviewers' comments, we have modified the Parallume materials so as to allow excitation with a longer wavelength of excitation light (365nm) which (a) prevents the photochemical decomposition of the DNA during optically decoding the samples, (b) allows non-UV optics to be used to focus the light and (c) lets much brighter, longer lived and less expensive LED light sources be used. A new surface chemistry, which strongly binds the DNA to the bead until it is chemically released, will provide yet another level of sample contamination protection. The improved MGM described here represents the first substantial progress since inception of the field in addressing the rapidly expanding storage requirements for DNA and nucleic acids. A new storage paradigm which employs optically encoded bead technology provides a sample density, degree of automation and simplicity not currently possible and with scalability to successfully store and retrieve any number of DNA samples for the foreseeable future. PUBLIC HEALTH RELEVANCE: In recent years there has been a very large increase in the number of samples of genetic materials such as DNA that have been synthesized or isolated from organisms. Some of these samples, such as an individual's DNA, forensic samples or genetic material from a new strain of bacteria are one-of-a-kind and cannot be replaced. However, there is currently no means to organize or automatically archive and retrieve DNA samples in an automatic and high throughput manner. Because of the increase in throughput from laboratories, governments, hospitals and universities, the problem is only expected to grow in the future. We offer here a design of a new instrument platform designated as the Microrepository for Genetic Materials (MGM). The samples will be stored in arrays of porous glass beads with each of the beads displaying an optical code or signature that uniquely identifies that particular sample and the DNA within that bead. The DNA samples are each placed into a single highly porous bead with a diameter of 50 microns (0.05mm). The beads are optically encoded with our ParallumeTM technology which allows each bead to be identified by the intensity ratios of the different colors emitted by the bead upon excitation. Thus, thousands of beads can be mixed together, each identified by its unique optical code and removed from the storage array using an optical fiber to pick it up. After checking the optical signature by examining the emitted light when the bead is picked up by the optical fiber, the bead can be placed in the desired destination location. Thus, we are able to design and build a repository that can store hundreds of thousands of DNA samples in a robust and inexpensive manner. This MGM technology is completely scalable and should provide rapid and accurate DNA storage and retrieval capacity for the foreseeable future.