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EASI--enrichment of alternatively spliced isoforms.

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Venables JP,Burn J

doi

10.1093/nar/gkl592

subject

Has Abstract

pub_date

2006-01-01 00:00:00

pages

e103

issue

15

eissn

0305-1048

issn

1362-4962

pii

gkl592

journal_volume

34

pub_type

杂志文章

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