Published online 1 September 2006 Nucleic Acids Research, 2006, Vol. 34, No. 15 e103 doi:10.1093/nar/gkl592 EASI—enrichment of alternatively spliced isoforms Julian P. Venables* and John Burn Institute of Human Genetics, International Centre for Life, Central Parkway, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, UK Received May 25, 2006; Revised July 27, 2006; Accepted August 1, 2006 ABSTRACT 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. INTRODUCTION The deﬁnition of alternatively spliced isoforms is ‘transcripts from the same gene that have identical ends with at least one tract of internal difference’. The principle of enrichment of alternatively spliced isoforms from cDNA relies on these alternative transcripts forming heteroduplexes, in which the missing region of one strand causes the ‘extra’ region in the other strand to form a single-stranded bulge, which can bind to a single-stranded DNA trap. Two groups have so far reported use of this method; in both cases to enrich alternatively spliced isoforms from complex cDNA libraries (1,2). Because of the ambitious nature of their task both pro- tocols contain complex denaturation and renaturation steps. Watahiki et al. (2) take full length cDNA libraries and tran- scribe them in vitro and then reverse-transcribe them again, just for their starting material. Thill et al. (1) perform three cycles of a procedure consisting of four different enzymatic steps per cycle to achieve a ﬁnal 10-fold enrichment of alternatively spliced isoforms. Although both groups demon- strate modest global enrichment of alternatively spliced isoforms, the vast scale of cellular alternative splicing means that they are currently unable to approach saturat- ing coverage at the individual gene level. Here we report a simple protocol (EASI) that enriches all the signiﬁcant alternatively spliced isoforms of individual target genes from ‘invisibility’ on agarose gels. As this enrichment pro- cedure adds just one hour’s work to current cloning methods it should be widely applicable in the study of alternative splicing. MATERIALS AND METHODS Making EASI beads Genomic DNA was isolated from Thermus aquaticus (YT1 strain from the centre for applied microbiology and research, Porton Down, Salisbury) and the single-stranded DNA- binding protein reading frame was ampliﬁed with the primers AAAAAAAACATATGGCTCGAGGCCTGAACCAA and AAAAAAAAGGATCCTCAAAACGGCAAATCCTCCTCC- GGCGGGAA and cloned into the NdeI and BamHI sites of PET15b (Novagen) to make the plasmid pEASI. pEASI was then transformed into Escherichia coli BL21. 0.5 mM IPTG was added to a 400 ml exponential culture growing in the presence of 100 mM ampicillin. After three further hours at 37 C, cells were harvested by centrifugation and resuspended in 5 ml of phosphate-buffered saline (PBS) and repeatedly sonicated (3 · 20 s) without cooling and then heated to 70 C (to precipitate E.coli proteins) for 5 min, then micro-centrifuged at top speed for 10 min. The supernatant was frozen in 400 ml aliquots and stored at 80 C. When needed, aliquots were thawed and further cleared by micro-centrifugation for 3 min and 80 ml (for each batch of lysate the saturating amount was determined by releasing bound protein from beads with Bradford reagent) Dynabeads TALON slurry (Dynal, Oslo) was washed in PBS and added to the supernatant and left on the bench for 5 min. The beads were magnetically separated and then incubated in (1 M NaCl and 20 mM Tris, pH 7.5) for 5 min to dissociate sheared E.coli DNA, separated again and then resuspended in 800 ml of PBS and stored in the fridge for use. The charged ssb-beads are stable indeﬁnitely at 4 C. *To whom correspondence should be addressed. Tel: +44 191 241 8636; Fax: +44 191 241 8666; Email j.venables@ncl.ac.uk 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.