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EASI - Enrichment of alternatively spliced isoforms

Lookup NU author(s): Dr Julian Venables, Professor Sir John Burn

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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. © 2006 Oxford University Press.


Publication metadata

Author(s): Venables JP, Burn J

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2006

Volume: 34

Issue: 15

Print publication date: 01/09/2006

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/nar/gkl592

DOI: 10.1093/nar/gkl592

PubMed id: 16951290


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