A RecA-mediated exon profiling method.
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, Interleukin1beta, 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.
Hasegawa
Yuki
Y
Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, 1-7-22 Suehiro-cho Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Fukuda
Shiro
S
Shimokawa
Kazuro
K
Kondo
Shinji
S
Maeda
Norihiro
N
Hayashizaki
Yoshihide
Y
D017398:Alternative Splicing; D000818:Animals; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D005091:Exons; D020869:Gene Expression Profiling; D007375:Interleukin-1 / Q000235:genetics; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D011938:Rec A Recombinases
D018076:DNA, Complementary; D007375:Interleukin-1; D015345:Oligonucleotide Probes; C051802:triplex DNA; D004247:DNA; D011938:Rec A Recombinases