Systematic analysis of mRNA 5' coding sequence incompleteness in Danio rerio: an automated EST-based approach

• Published in: Biology direct Vol. 2; no. 1; p. 34
• Main Authors: Frabetti, Flavia, Casadei, Raffaella, Lenzi, Luca, Canaider, Silvia, Vitale, Lorenza, Facchin, Federica, Carinci, Paolo, Zannotti, Maria, Strippoli, Pierluigi
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 27.11.2007; BioMed Central; BMC
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cloning; Cloning, Molecular; Cluster Analysis; Computational Biology; Databases, Factual; DNA, Complementary - genetics; Expressed Sequence Tags; Genetic aspects; Messenger RNA; Molecular Sequence Data; Open Reading Frames - genetics; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Software; Zebra fish; Zebrafish - genetics; Zebrafish Proteins - genetics; United States
• DOI: 10.1186/1745-6150-2-34
• ISSN: 1745-6150

All standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish). We implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR). The implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish.