In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs

Abstract

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-m 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 Ϸ10 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.DNA analysis ͉ gene expression ͉ parallel cloning ͉ fluid microarray

Writing Information into DNA

“…3 All of the strong constraints could be satisfied with only 3 bases [15]. Other groups that employed three-base words likewise used random word-generation for their word design [24,23].…”

“…To integrate the terminal bases, we assume as if x is cyclic in the computation of frequency. For example, AAGCGCTT and TACGGCAT exhibit close melting temperatures because they share the same dimer frequency (3,2,3). Thus, all DNA code words should share the same dimer frequency to guarantee their concerted behavior.…”

“…Typically, a set of fixed-length oligonucleotides is used to denote logical variables or graph components. -DNA tag/antitag system which designs fixed-length short oligonucleotide tags for identifying biomolecules (e.g., cDNA), used primarily for monitoring gene expressions [2,3,4]. -DNA data storage which advocates the use of bacterial DNA as a long-lasting high-density data storage, which can also be resistant to radiation [5].…”

Biological microbeads for flow‐cytometric immunoassays, enzyme titrations, and quantitative PCR

“…While this approach matches conventional methods in sensitivity, reproducibility, and simplicity, it seems to have significant advantages by virtue of the possibility for multiplex analysis (5,6,9,(11)(12)(13)(14)(15). Furthermore, sequence-specific capture of PCR-amplified genomic or cDNA sequences allows the detection of single nucleotide polymorphisms (16)(17)(18), and also turns this platform into a possible alternative to microarrays on chips to be used for the characterization of gene expression profiles (19).…”

Improved picoliter-sized micro-reactors for high-throughput biological analysis

“…However, the reliance on capillary electrophoresis means that conventional sequencing is not likely to become a particularly low-cost, high-speed, or high-throughput technique. The advent of next-generation sequencing, based on massively parallel arrays of individual sequencing reactions, has drastically improved the sequencing throughput and greatly reduced cost and time [2][3][4][5][6][7]. High-throughput pyrosequencing [8], relying on detection of chemiluminescence signals generated from millions of DNA polymerization reactions, has shown excellent accuracy, with longer read lengths than those available with other next-generation technologies, and has been successfully used for de novo sequencing, resequencing, and metagenomic sequencing [9][10][11][12][13].…”