Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons.
We herein describe an integrated system for the high-throughput analysis of splicing events and the identification of transcript variants. The system resolves individual splicing events and elucidates transcript variants via a pipeline that combines aspects such as bioinformatic analysis, high-throughput transcript variant amplification, and high-resolution capillary electrophoresis. For the 14 369 human genes known to have transcript variants, minimal primer sets were designed to amplify all transcript variants and examine all splicing events; these have been archived in the ASprimerDB database, which is newly described herein. A high-throughput thermocycler, dubbed GenTank, was developed to simultaneously perform thousands of PCR amplifications. Following the resolution of the various amplicons by capillary gel electrophoresis, two new computer programs, AmpliconViewer and VariantAssembler, may be used to analyze the splicing events, assemble the consecutive exons embodied by the PCR amplicons, and distinguish expressed versus putative transcript variants. This novel system not only facilitates the validation of putative transcript variants and the detection of novel transcript variants, it also semi-quantitatively measures the transcript variant expression levels of each gene. To demonstrate the system's capability, we used it to resolve transcript variants yielded by single and multiple splicing events, and to decipher the exon connectivity of long transcripts.
Hsiao
Tzu-Hung
TH
Departmant of Electrical Engineering, National Taiwan University, Taipei, Taiwan 106, ROC.
Lin
Chien-Hong
CH
Lee
Te-Tsui
TT
Cheng
Ji-Yen
JY
Wei
Pei-Kuen
PK
Chuang
Eric Y
EY
Peck
Konan
K
D017398:Alternative Splicing; D019295:Computational Biology; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D005091:Exons; D014644:Genetic Variation; D006801:Humans; D020407:Internet; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry; D014158:Transcription, Genetic; D014584:User-Computer Interface
D017931:DNA Primers; D012333:RNA, Messenger
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