Brenner S, Williams S R, Vermaas E H, Storck T, Moon K, McCollum C, Mao J I, Luo S, Kirchner J J, Eletr S, DuBridge R B, Burcham T, Albrecht G
Lynx Therapeutics, Inc., 25861 Industrial Boulevard, Hayward, CA 94545, USA.
Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1665-70. doi: 10.1073/pnas.97.4.1665.
We describe a method for cloning nucleic acid molecules onto the surfaces of 5-micrometer microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry approximately 10(6) strands complementary to one of the tags. About 10(5) copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.
我们描述了一种将核酸分子克隆到5微米微珠表面而非生物宿主中的方法。每个分子都连接有一个独特的标签序列，并且对带有标签的文库进行扩增。通过从非常大量的标签序列库中抽取一小部分（1%）来实现分子的独特标记。所得文库与微珠杂交，每个微珠携带大约10⁶条与其中一个标签互补的链。每个微珠上收集大约10⁵个每个分子的拷贝。由于这些克隆在微珠上是分离的，它们可以同时进行操作，然后分别进行检测。为了证明这种方法的实用性，我们展示了如何通过使用荧光激活细胞分选仪（FACS）对携带在两个文库之间差异表达的克隆的微珠进行标记和提取。因为不需要关于克隆分子的先验信息，所以在序列数据库不完整或不存在的情况下，这个过程显然是有用的。更重要的是，这个过程还允许分离仅在给定组织中表达或在正常和疾病状态之间差异表达的克隆。然后可以将这些克隆点样到更具成本效益的、针对组织或疾病的低密度平面微阵列上。
