High Throughput Microrepository for Genetic Materials

描述（由申请人提供）：目前的许多技术正在越来越困难地解决对医疗、法医和研究应用中遗传材料储存和检索的安全、快速和可靠技术的预期需求。我们提出了一种新型的可扩展的微储存库，其样品制备，鉴定和检索协议提供了一个即时的和实质性的改进，在几个重要的方面，现有的储存库技术，并可以配置为提供一个自我补充功能的存储的遗传物质。遗传物质微储存库（MGM）是基于光学编码珠，硅微加工，机械珠处理和可选的珠上PCR的组合，以取代从储存库中取出的遗传物质。给定的DNA样品被储存在多孔玻璃或聚合物珠中，该珠具有其自己独特的光学代码。这些珠子是用稀土纳米颗粒编码的，可以产生多达六个可分辨的可见光发射波段，并可以支持数千个可分辨的光学特征。将合并的编码珠随机加载到微加工的硅珠保持夹具中，该夹具提供数千个光学隔离的威尔斯，并且根据其珠的光学特征确定给定DNA样品在保持器内的位置。使用来自合作者BioDot，Inc.的先进珠处理技术，从硅存储板上的x，y坐标中识别的珠或珠浆被取出并置于目的位置，可能是PCR板。如果储存材料的序列的一部分是已知的，则储存珠粒可以含有与珠粒共价结合的引物。已经表明，有效的PCR扩增可以在珠的孔内非常快速地发生，得到的扩增子通过引物共价结合到珠上。扩增后，通过简单地使dsDNA变性并允许解离的单链扩散出珠并进入溶液中，珠上的双链（ds）DNA可用于扩增溶液中的更多材料，在溶液中其可用作PCR的模板。由于一条链始终保持共价连接到珠上，因此珠可以被替换到原始储存位置，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 nm的刚性孔中，细菌无法进入孔中，因此可以在室温下储存一些样品。

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.