Massively massive parallel analysis

On page 630, Brenner et al. present a clever technology for sequencing 20-nucleotide-long stretches of genes–hundreds of thousands at a time. As a tool for global analysis of gene expression, the technique, called massively parallel signature sequencing (MPSS), may give DNA microarrays and “clone and count” methods like SAGE a run for their money. They started by constructing a cDNA library on the surface of a million microbeads, which they then immobilized in a flow chamber, where a series of solutions could wash over them, taking them through a series of cycles that make up the novel sequencing reaction. First an adapter sequence is ligated to the cDNA. It contains the binding site for BbvI, a restriction enzyme that can reach over to the cDNA some distance from its binding site, and cleave it to expose a short bit of single-stranded sequence. Next, a single-stranded adapter binds to its complementary exposed sequence, followed by a set of fluorescent decoder oligonucleotides that reveal the identity of the bit of the cDNA sequence as the microbead flies past a detector. This is repeated for five cycles, providing enough sequence to identify the cDNA on each bead in the collection. The more a cDNA is represented, the more abundant is its expression in the cells from which the library was made. In addition to its impressively high throughput, MPSS has the advantages of detecting extremely rare genes, not requiring prior knowledge of gene sequences, and providing a statistically robust digital output. (See also p. 597)

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DeWitt, N. Massively massive parallel analysis. Nat Biotechnol 18, 586 (2000). https://doi.org/10.1038/76379

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