Enrichment of Alternatively Spliced Isoforms

Most metazoan genes are alternatively spliced, and a large number of alternatively spliced isoforms are likely to be functionally significant and expressed at specific stages of pathogenesis or differentiation. Splicing changes usually only affect a small portion of a gene, and these changes may cause significant mRNA degradation. After RT–PCR, minor variants can form heteroduplexes with the major variants. Affinity purification of these heteroduplexes using immobilized Thermus aquaticus single-stranded DNA-binding protein allows purification of alternative splice forms in a 1:1 ratio, which makes it easy to sequence the rare form. This chapter provides a detailed protocol of the technique I have developed to identify spliced isoforms called enrichment of alternatively spliced isoforms or EASI.