High Throughput Microrepository for Genetic Materials

遗传物质高通量微存储库

• 批准号: 7482531
• 负责人: ROBERT C HAUSHALTER
• 金额: $ 12.07万
• 依托单位: PARALLEL SYNTHESIS TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7482531
• 关键词: Address; Archives; Arts; Back; Bacteria; Binding; Biological Preservation; Caliber; Cells; Code; Collaborations; Computer software; Cryopreservation; DNA; DNA Probes; DNA Sequence; Databases; Destinations; Detection; Development; Diffuse; Educational workshop; Emerging Technologies; Enzymes; Forensic Medicine; Freezing; Future; Genetic Materials; Glass; Goals; Hour; Individual; Instruction; Label; Lasers; Liquid substance; Location; Measures; Mechanics; Medical; Modification; Movement; National Center for Research Resources; Numbers; Operative Surgical Procedures; Optical Methods; Optics; Other Genetics; Output; Phase; Polymerase Chain Reaction; Polymers; Preparation; Process; Protocols documentation; RNA; Rate; Reading; Recommendation; Reporting; Research; Retrieval; Risk; Sampling; Scheme; Silicon; Solutions; Speed; System; Technology; Temperature; Time; United States National Institutes of Health; Writing; base; density; design; desire; ds-DNA; egg; gel electrophoresis; nanoparticle; new technology; novel; prevent; prototype; repository; research study; size

DESCRIPTION (provided by applicant): The projected demands for safe, rapid and reliable technology for the storage and retrieval of genetic materials in medical, forensic and research applications are being addressed with increasing difficulty by many of the current technologies. We propose a new type of scalable microrepository whose sample preparation, identification and retrieval protocols offer an immediate and substantial improvement over the existing repository technologies in several important ways and can be configured to provide a self-replenishing feature for the stored genetic materials. The Microrepository for Genetic Materials (MGM) is based on a combination of optically encoded beads, silicon micromachining, mechanical bead handling and, optionally, on-bead PCR to replace the genetic material removed from the repository. A given sample of DNA is stored in a porous glass or polymer bead which possesses its own unique optical code. The beads are encoded with rare earth-based nanoparticles, which produce up to six resolvable visible emission bands, and can support thousands of resolvable optical signatures. The pooled encoded beads are randomly loaded into a micromachined silicon bead-holding fixture, which provides thousands of optically isolated wells, and the location of a given DNA sample within the holder determined from the optical signature of its bead. Using advanced bead handling technology from collaborator BioDot, Inc., a bead or bead slurry, identified from its x,y coordinates on the silicon storage plate is removed and placed into the destination location, presumably a PCR plate. If a portion of the sequence of the stored material is known, then the storage beads can contain a primer covalently bound to the bead. It has been shown that efficient PCR amplification can take place very rapidly within the pores of the bead with the resulting amplicon covalently bound to the bead via the primer. After amplification, the double stranded (ds) DNA on the bead can be used to amplify more material in solution by simply denaturing the dsDNA and allowing the dissociated single strand to diffuse out of the bead and into solution where it can be used as the template for PCR. Since one strand always remains covalently attached to the bead, the bead can be replaced into the original storage location ready for a future PCR amplification thereby providing a perpetually filled repository. Since the DNA is stored in rigid pores <300 nm in diameter, bacteria can not enter the pores and it may be possible to store some samples at room temperature.

描述(由申请人提供)：在医疗、法医和研究应用中，对储存和检索遗传物质的安全、快速和可靠技术的预期需求正在通过许多现有技术越来越困难地得到满足。我们提出了一种新型的可扩展的微库，其样品准备、鉴定和检索协议在几个重要方面比现有的库技术提供了立即和实质性的改进，并且可以被配置为为存储的遗传物质提供自我补充的特征。遗传材料微库(MGM)是基于光学编码微珠、硅微加工、机械微珠处理和可选的微珠聚合酶链式反应(on-bead PCR)的组合，以取代从储存库中移除的遗传物质。给定的DNA样本储存在多孔玻璃或聚合物珠中，它们拥有自己独特的光学代码。这些珠子是用基于稀土的纳米颗粒编码的，可以产生多达六个可分辨的可见发射波段，并可以支持数千个可分辨的光学特征。将汇集在一起的编码珠随机加载到微机械硅珠夹具中，该夹具提供数千个光学隔离的孔，并根据珠子的光学签名确定给定DNA样本在夹持器中的位置。使用来自合作者BioDot，Inc.的先进的珠粒处理技术，从其在硅储存板上的x，y坐标识别的珠粒或珠浆被移除并放置到目的地位置，假设是PCR板。如果存储材料的序列的一部分是已知的，则存储珠可以包含共价结合到珠上的引物。已经证明，高效的PCR扩增可以在珠子的毛孔内非常迅速地进行，所得到的扩增产物通过引物共价结合到珠子上。扩增后，小球上的双链DNA可以用来在溶液中扩增更多的物质，方法是简单地变性双链DNA，并允许解离的单链从小球扩散到溶液中，在那里它可以用作聚合酶链式反应的模板。由于一条链始终保持与珠子的共价连接，因此可以将珠子替换到原始存储位置，为将来的PCR扩增做好准备，从而提供永久填充的储存库。由于DNA储存在直径300纳米的硬质小孔中，细菌不能进入小孔，因此可以在室温下储存一些样品。