EASI--enrichment of alternatively spliced isoforms.
Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts.
Venables
Julian P
JP
Institute of Human Genetics, International Centre for Life, Central Parkway, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK. j.venables@ncl.ac.uk
Burn
John
J
D017398:Alternative Splicing; D001709:Biotechnology / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D013824:Thermus / Q000235:genetics
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins