e103 Nucleic Acids Research, 2006, Vol. 34, No. 15 its efﬁciency and usefulness. The T.Aq ssb column described in this work can be easily prepared from recombinant bacteria using heat to precipitate all host E.coli proteins without fear of denaturing the thermostable ssb. No complex protein puriﬁcation is needed, as by mixing beads with the heat- cleared lysate the His-tagged protein can thus be stored bound to beads and ready for use for >3 months with no loss of activity (data not shown). As Talon-Dynabeads have a far smaller size than other nickel-agarose beads they have the advantage that no complex chromatography steps are needed in the enrichment as the beads stay suspended and effectively ‘in solution’ for the course of the binding reaction. Binding to the ssb-bead suspension is quick and extremely speciﬁc, as single-stranded DNA binding was unaffected by a 100-fold excess of double stranded DNA (data not shown). The recently discovered T.Aq ssb may perform better than E.coli ssb because it is a tandem dimer and does not need to tetramerize to become active (6). Similarly, tandem dimer red ﬂuorescent protein has been shown to have enhanced kinetics of activation compared to the normal tetrameric form (7). The ﬁnal cloning procedure also contains several time saving steps compared to the previous methods. To avoid restriction digestion, EASI material is directly ligated into a TA cloning vector and transformed into E.coli. Colony-PCR then allows all the alternative splice forms in the sample to give high quality sequence. This avoids the process of gel puriﬁcation of bands that include heteroduplex bands that are difﬁcult to clone and could lead to redundancy (8). The development of a simple protocol for enrichment of alternatively spliced isoforms from PCRs represents a signiﬁcant step forward from previous methods; in one method ‘invisible’ isoforms were rescued by PCR ampliﬁca- tion of all the sections of agarose gel lanes that did not contain any visible bands (9). Our simple and robust approach takes the labour and chance out of alternative splicing discovery and will allow the prevalence and biological signiﬁcance of splicing events to be fully explored. For example in testis there are several cell types: Sertoli, Leydig and germ cells. The germ cell population consists of cells in various stages of differentiation from stem cell to cells before and during meiosis to condensing haploid cells. It is thus difﬁcult to ﬁnd alternative splices in the stem cells as they constitute <10% of the tissue mass. The same problem applies to cancer stem cells in a tumour mass. Therefore with EASI it should be possible to deﬁne a limited set of PAGE 4 OF 4 all the alternative splices in a healthy or diseased tissue in order to study cell type speciﬁc splicing regulation. In addition we are proposing to develop EASI for high- throughput clinical diagnostics in the hope of identifying mutations that affect splicing responsible for genetic disease, especially in cases where the resulting isoforms are rendered ‘invisible’ in agarose gels by nonsense mediated decay. ACKNOWLEDGEMENTS This work was funded by the Newcastle Healthcare Charity. Funding to pay the Open Access publication charges for this article was provided by Newcastle Healthcare Charity. Conflict of interest statement. None declared. REFERENCES 1. Thill,G., Castelli,V., Pallud,S., Salanoubat,M., Wincker,P., de la Grange,P., Auboeuf,D., Schachter,V. and Weissenbach,J. (2006) ASEtrap: a biological method for speeding up the exploration of spliceomes. Genome Res., 16, 776–786. 2. 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