High Throughput Microrepository for Genetic Materials

描述（由申请人提供）：认识到迫切需要一种安全、快速和可靠的方法来保存和存档遗传样本和其他生物材料，如合成DNA样本、独特分离样本、质粒、法医样本、患者样本、确保植物或动物未来生物多样性的样本、克隆和考古样本，最近由NIH NCRR召集的一个研究小组确定了最迫切需要的领域，并提出了一些建议。在他们的主要建议清单中，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低温保存技术”和“支持新的‘高风险/高回报’保存技术，这些技术将开辟新的领域”。我们已经直接并成功地解决了这些需求，我们的遗传材料微库（MGM）技术提供了完全可扩展的，非常高密度的样品存储，通过在单一平台上提供样品制备-识别-存档-检索功能，从光复用编码珠阵列中自动检索。在第一阶段的工作中，一个完全自动化的米高梅平台原型成功构建并进行了测试。米高梅包含密集的多孔玻璃或聚合物珠阵列，小珠组或纸样，每个包含不同的DNA样本，这些样本已经用我们基于稀土的平行技术进行了光学编码，使每个珠子都能被唯一地识别出来。将DNA放置到单个大珠子上后，将珠子装入珠子定位载玻片（BLS），该载玻片将珠子光学隔离成平面单层以进行成像，并读取每个珠子的光学代码以确定特定DNA样本的位置。这些珠子被取出并放置在所需的位置，并且在处理过程中通过qPCR确认没有污染。通过进行额外的实验，包括结合新的改进硬件设计和成功地改变Parallume编码材料的化学成分，以防止紫外线对DNA的损害，我们解决了所有审稿人的担忧。我们现在已经建立并成功测试了一个功能齐全的MGM仪器原型，并且根据评论者的意见，我们修改了平行材料，以便使用更长的激发光波长（365nm）进行激发，这可以(a)防止在光学解码样品时DNA的光化学分解，(b)允许使用非紫外光学元件来聚焦光，(c)让更亮的。使用寿命更长、成本更低的LED光源。一种新的表面化学，将DNA与样品紧密结合，直到DNA被化学释放，将提供另一种水平的样品污染保护。这里描述的改进的MGM代表了自该领域成立以来在解决DNA和核酸快速扩展的存储需求方面的第一个实质性进展。一种采用光学编码磁珠技术的新存储模式提供了目前无法实现的样品密度、自动化程度和简单性，并且具有可扩展性，可以在可预见的未来成功存储和检索任意数量的DNA样品。

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.