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