This Article Full Text Full Text (PDF) Supplemental Research Data All Versions of this Article: gr.5063306v1 16/6/776    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thill, G. Articles by Weissenbach, J. Search for Related Content PubMed PubMed Citation Articles by Thill, G. Articles by Weissenbach, J. Social Bookmarking                   What's this?

Methods

ASEtrap: A biological method for speeding up the exploration of spliceomes

¹ Genoscope-Centre National de Séquençage and Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR)-8030, 91000 Evry, France; ² Institut National de la Santé et de la Recherche Médicale (INSERM) U685/AVENIR, Centre G. Hayem, Institut Universitaire d'Hématologie, Hôpital Saint Louis, 75010 Paris, France

Alternative splicing (AS) of pre-messenger RNA is a major mechanism for generating protein diversity from a limited number of genes in higher eukaryotes, and it constitutes a central mode of genetic regulation. Thus, efficient methods are needed to systematically identify new AS events at a genomic scale across different tissues, stages of development, and physiological or pathological conditions in order to better understand gene expression. To fulfill this goal, we have designed the ASEtrap, which is a cloning procedure for producing AS libraries that is based on a single-stranded trap consisting of an ssDNA-binding protein. In this paper, we have applied our approach to the construction of an AS library and a Control library from human placenta. By analyzing 9226 and 9999 sequences of the AS and Control libraries, respectively, we show that internal AS events (events that can be identified by the sole resources provided by either the AS or the Control library) and the discovery rate of new AS events measured at early stages of sequencing were nine to 10 times higher in the former than in the latter. Moreover, by performing a search for new AS events within a group of 162 known drug target genes, we identified six new events in six genes, and we observed that they all were discovered exclusively through the AS library. Thus, it appears that ASEtrap has the potential to greatly facilitate the determination of the total complement of splice variants expressed in human, as well as other organisms.

E-mail thill-asplicing{at}genoscope.cns.fr; fax 33-01-60-87-25-14.

[Supplemental material is available online at www.genome.org.]

Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5063306

⁴ The average size of the double-stranded cDNA molecules obtained at the end of the preparation step was 1.5 kb (data not shown); consequently, the transcripts expressed in placenta should be, on average, represented by about five to six different RsaI restriction fragments in the Control library. The frequency in placenta of a transcript not represented by any ESTs from the Control library is therefore bound to be less than 5/9999–6/9999, that is, 5 x 10^–4 to 6 x 10^–4.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				P. Carninci Constructing the landscape of the mammalian transcriptome J. Exp. Biol., May 1, 2007; 210(9): 1497 - 1506. [Abstract] [Full Text] [PDF]

				J. P. Venables and J. Burn EASI--enrichment of alternatively spliced isoforms Nucleic Acids Res., September 10, 2006; 34(15): e103 - e103. [Abstract] [Full Text] [PDF]