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


RESULTS
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).


CONCLUSIONS
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.
Frabetti
Flavia
F
Center for Research in Molecular Genetics "Fondazione CARISBO", Department of Histology, Embryology and Applied Biology, University of Bologna, via Belmeloro 8, 40126 Bologna (BO), Italy. flavia@alma.unibo.it
Casadei
Raffaella
R
Lenzi
Luca
L
Canaider
Silvia
S
Vitale
Lorenza
L
Facchin
Federica
F
Carinci
Paolo
P
Zannotti
Maria
M
Strippoli
Pierluigi
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D016208:Databases, Factual; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012984:Software; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D029961:Zebrafish Proteins