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Nucleic Acids Research Advance Access originally published online on September 1, 2006
Nucleic Acids Research 2006 34(15):e103; doi:10.1093/nar/gkl592
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Nucleic Acids Research, 2006, Vol. 34, No. 15 e103
© 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.

Methods Online

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

*To whom correspondence should be addressed. Tel: +44 191 241 8636; Fax: +44 191 241 8666; Email j.venables{at}ncl.ac.uk

Received May 25, 2006. Revised July 27, 2006. Accepted August 1, 2006.

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.


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