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.
Venables
Julian P
JP
Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke Centre de développement des biotechologies (CDB) de Sherbrooke, Québec, Canada.
D017398:Alternative Splicing; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008297:Male; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D013824:Thermus / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D012333:RNA, Messenger; D011994:Recombinant Proteins