20] Hybrid selection of rnRNA with biotinylated DNA

Abstract

This chapter discusses the procedure to make the biotinylated single-stranded DNA with the use of an asymmetric polymerase chain reaction (PCR) is described for use in a high-efficiency hybrid selection procedure. After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA-DNA onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The hybrid selection method with ssDNA-B has several advantages over the filter-binding techniques. The asymmetric PCR with excess of biotinylated primer makes it possible to generate the ssDNA-B, which has a biotin molecule with an extended arm that in turn facilitates the binding of the hybrids to the streptavidin agarose without steric hindrance. In addition, ssDNA-B can be used in solution hybridization, which is more rapid and efficient than filter hybridization. This procedure can be utilized in various additional applications, such as selecting a set of mRNAs with a fragment of DNA with complementary sequence, enrichment of homologous mRNA to the probe DNA-B, and for hybrid depletion assays.