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Nucleic Acids Research 2006 34(13):e97; doi:10.1093/nar/gkl497
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Published online 8 August 2006

Nucleic Acids Research, 2006, Vol. 34, No. 13 e97
© 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-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A RecA-mediated exon profiling method

Yuki Hasegawa1,2, Shiro Fukuda1, Kazuro Shimokawa1, Shinji Kondo1, Norihiro Maeda3 and Yoshihide Hayashizaki1,2,3,*

1 Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute 1-7-22 Suehiro-cho Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 2 International Graduate School of Arts and Sciences, Yokohama City University 1-7-29 Suehiro-Cho, Tsurumi-Ku, Yokohama 230-0045, Japan 3 Genome Science Laboratory, Discovery and Research Institute RIKEN Wako Main Campus, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

*To whom correspondence should be addressed. Tel: +81 45 503 9222; Fax: +81 45 503 9216; Email: rgscerg{at}gsc.riken.jp

Received June 9, 2006. Revised June 28, 2006. Accepted June 29, 2006.

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1ß, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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