Karen L. Fearon

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3 × 106 microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16–20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Synthesis and purification of oligonucleotide N3'-->P5' phosphoramidates and their phosphodiester and phosphorothioate chimeras.

This unit describes the synthesis and purification of oligonucleotide N3′→︀P5′ phosphoramidates, wherein each 3′‐oxygen is replaced by a 3′‐amine in the 2′‐deoxyribose ring. The synthesis of required monomers and application of the method to preparation of phosphodiester‐ and phosphorothioate‐containing chimera of phosphoramidate is also reported.