High Throughput Microrepository for Genetic Materials

描述(申请人提供)：高吞吐量遗传材料微库NIH NCRR最近召集的一个研究小组认识到迫切需要一种安全、快速和可靠的手段来保存和存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、确保未来植物或动物、克隆和考古样本的生物多样性的样本，因此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项和第五项是“鼓励发展高通量和可扩展的种质加工和超低温保存技术”，以及“支持新的‘高风险/高回报’的保存技术，这将是新的突破”。我们的遗传材料微库(MGM)技术直接并成功地满足了这些需求，该技术通过在单一平台上提供样本preparation-identification-archiving-retrieval功能，提供完全可扩展的、非常高密度的样本存储，以及从光学多路复用编码珠数组中自动检索。在第一阶段的工作中，成功地建造了一个完全自动化的米高梅原型平台并进行了测试。米高梅含有致密的多孔玻璃或聚合物微珠阵列、一组较小的微珠或纸样，每个微珠或纸样包含一个不同的DNA样本，这些样本已使用我们基于稀土的Parallume技术进行光学编码，从而使每个微珠能够被唯一地光学识别。在将DNA放置在单个大珠子上后，将珠子加载到珠子定位载玻片(BLS)中，该载玻片将珠子光学隔离成平面单层用于成像，并读取每个珠子的光学代码以确定该特定DNA样本的位置。珠子被取回并放置在所需的位置，并且通过定量聚合酶链式反应证实在处理过程中没有污染。通过进行其他实验，包括采用新的改进的硬件设计，并成功地改变Parallume编码材料的化学成分以防止紫外线对DNA的损害，我们已经解决了所有审查者的担忧。我们现在已经建造并成功测试了一个功能齐全的MGM仪器原型，为了回应有洞察力的评审员的意见，我们已经修改了Parallume材料，以便能够用更长波长(365 Nm)的激发光进行激发，从而(A)防止在对样品进行光学解码过程中DNA的光化学分解，(B)允许使用非紫外光来聚焦光线，(C)让LED光源更明亮、更长寿命、更便宜。一种新的表面化学将DNA强烈结合到珠子上，直到它被化学释放，这将提供另一种水平的样本污染保护。这里描述的改进的米高梅是该领域成立以来在解决迅速扩大的DNA和核酸存储需求方面取得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目前无法实现的样本密度、自动化程度和简单性，并且具有在可预见的未来成功存储和检索任意数量的DNA样本的可扩展性。 与公共卫生相关：近年来，从生物体中合成或分离的DNA等遗传物质的样本数量有了很大的增加。其中一些样本，如个人的DNA、法医样本或来自新菌株的遗传物质，是独一无二的，不可替代。然而，目前还没有办法以自动和高通量的方式组织或自动存档和检索DNA样本。由于来自实验室、政府、医院和大学的吞吐量增加，预计未来这个问题只会越来越严重。我们在这里提供了一个新的仪器平台的设计，被指定为遗传材料微库(MGM)。样本将储存在多孔玻璃微珠阵列中，每个微珠显示一个光学代码或签名，唯一地识别该特定样本和该微珠中的DNA。每个DNA样本都被放入一个直径为50微米(0.05毫米)的高度多孔的珠子中。珠子使用我们的ParallumeTM技术进行光学编码，该技术允许根据珠子在激发时发出的不同颜色的亮度比来识别每个珠子。因此，数以千计的珠子可以混合在一起，每个珠子由其唯一的光学代码识别，并使用光纤将其从存储阵列中移除。在通过检查当珠子被光纤拾取时发射的光来检查光学特征之后，可以将珠子放置在期望的目的地位置。因此，我们能够设计和建立一个可以以健壮和廉价的方式存储数十万个DNA样本的存储库。这项米高梅技术是完全可扩展的，在可预见的未来应该可以提供快速和准确的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.