Lynx Therapeutics (Hayward, CA) combines two techniques to assess gene expression: the ability to sequence without using a gel, and the ability to clone millions of templates onto microbeads . Specifically, cDNA templates are first ligated into vectors containing a set of different 32-mer oligonucleotide tags. A 1% subset of these ligated vectors is taken, such that each template is ligated to a unique tag and each template is represented at least once. The subset is then PCR-amplified, made single-stranded, then hybridized against a set of microbeads each separately containing a sequence complementary against a 32-mer tag. The microbeads are then fixed in place within a flow cell. Sequencing can take place directly on the microbeads through successive use of restriction enzymes exposing 4 base pairs, which in turn can be successively bound by florescently labeled probes.
Since the source templates can be generated from mRNA, this can effectively be used as a massively parallel method to find tags associated with each mRNA. Also, since presumably each copy of each mRNA is uniquely labeled with a different tag, each copy is sequenced separately, and thus this system is not only quantitative, but may be more sensitive at lower gene expression levels, like SAGE. At the time of writing, commercialization of this technique has just started.
Was this article helpful?