PAGE 3 OF 4 Nucleic Acids Research, 2006, Vol. 34, No. 15 e103 Figure 1. (a) EASI proof of principle. A known pair of alternative splices was bound to beads charged with E.coli or T.Aq ssb, then eluted, amplified by PCR and visualized by agarose electrophoresis. The alternatively spliced isoform of HipK3 (upper band) was enriched by the ssb beads. (b) Two overlapping sections of p53 were enriched after exonuclease treatment. Agarose gel showing two PCR product inputs to the EASI column (first lane on each side). The second lanes are a 10 000:1 dilution of the input subjected to a further 25 rounds of PCR (to show that enrichment is not a PCR artefact). Lanes 3, 4 and 5 of each side show the enriched output after applying three conditions: no treatment, addition of extra T.Aq polymerase for 10 min, or addition of Exonuclease VII. (c) Further confirmation. Three sections of MLH1 and four sections of MSH2 are shown. Each section shows input, diluted input PCR’d and EASI material in the third lane. The exons amplified in each section are indicated. (b and c). Sequences of the alternative splice forms found by EASI have been deposited in GenBank accession no. DQ648883–DQ648897. Arrows show alternative splice forms: (1) TP53+ins exon 1a, (2) TP53-Ex4, (3) TP53-Ex(2–4) (4) TP53ins9a (p53b), (5) TP53-Ex10, (6) MLH1ins ds 50ss 1a, (7) MLH1-Ex6, (8) MLH1-Ex10, (9) MLH1-Ex(9–10), (10) MLH1-Ex(15– 18), (11) MLH1-Ex(14–18), (12) MSH2-Ex3, (13) MSH2-Ex5, (14) MSH2+ins exon 9a, (15) MSH2-Ex10. better (to the theoretical maximum of 50%) so T.Aq ssb was used for all subsequent work. Our procedure was then used to discover new splice forms, from overlapping sections of the TP53 gene. Initially, two alternative splices were enriched in the 50 section but no enrichment was observed in the 30 end which was perplexing as there is a known alternative splice [p53insEx9a (4)] in the 30 end. Therefore we used two treatments to reduce single- stranded DNA contamination from the input PCRs in the hope of improving enrichment (Figure 1b). Adding fresh polymerase to the PCR, for 10 min at 72 C, before enrich- ment, slightly improved enrichment of the 50 end but had no effect on the 30 end. However digesting single-stranded DNA (in both directions) from the PCR with exonuclease VII improved the 50 result yet further, and signiﬁcantly it caused the appearance of the p53beta isoform in the 30 end. Exonuclease VII was therefore incorporated into the sub- sequent protocol (Materials and Methods). To consolidate these results, three overlapping sections of MLH1 and four sections of MSH2 were enriched (Figure 1c). In all, 15 extra bands appeared in the EASI fractions for the three tumour suppressor genes (deposited in GenBank accession nos DQ648883-97). Of these 15, only four were known previously; the TP53ins9a is also known as the beta isoform (4) and three others have been deposited as ESTs very recently: TP53-Ex(2–4) accession DA901020, MLH1+ins1a accession DA499490 and MLH1-Ex6 acces- sion DA343206 (5). The other 11 of these 15 sequences are unknown previously, validating the use of EASI for the discovery of new splice forms. DISCUSSION The EASI protocol described here contains several short-cuts from previously described methods that help to increase