High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper

Carninci, Piero; Kvam, Catrine; Kitamura, Akiko; Ohsumi, Tatsuya; Okazaki, Yasushi; Itoh, Mitsuteru; Kamiya, Mamoru; Shibata, Kazuhiro; Sasaki, Nobuya; Izawa, Masaki; Muramatsu, Masami; Hayashizaki, Yoshihide; Schneider, Claudio

doi:10.1006/geno.1996.0567

Cited by 283 publications

(216 citation statements)

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“…In strict sense, a full-length cDNA should cover both the ORF and the complete 5Ј and 3Ј UTR. Although a number of methods have been used to surmount the technical obstacles for getting the 5Ј end of cDNA (Carninci et al 1996), it is still difficult to reach the transcription start site in many cases. However, as the most important functional information of an mRNA is contained in the ORF, cDNAs containing entire ORFs are often considered as being full-length.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Functional Analysis of cDNAs with Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

Zhang

Yang

Wu³

et al. 2000

Genome Res.

162

127

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Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58-752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon-intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3Ј UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation.

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Section: Discussionmentioning

confidence: 99%

Cloning and Functional Analysis of cDNAs with Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

Zhang

Yang

Wu³

et al. 2000

Genome Res.

162

127

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“…Except for the library 01 ; Table 1, complete table at www.genome.org), the remaining 245 libraries were enriched for full-length cDNAs by Cap-Trapper (Carninci et al 1996Carninci and Hayashizaki 1999; Tables 1 and 3). Such libraries, represented in chronological order (Table 1, complete table at www.genome.org), include 390 sublibraries that represent different cDNA cloning events from the same starting mRNA and usually differ for cDNA preparation protocols, such as normalization, subtraction, cloning vector, or size selection.…”

Section: Multiple Strategies Are Necessary For Gene Discoverymentioning

confidence: 99%

“…The introduction of full-length cDNA libraries by Cap-Trapper or other technologies that take advantage of the peculiarity of the capstructure, (Carninci et al 1996Suzuki et al 1997;Carninci and Hayashizaki 1999;Mizuno et al 1999) did not solve this problem completely.…”

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confidence: 99%

Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

Carninci¹,

Waki²,

Shiraki³

et al. 2003

Genome Res.

Self Cite

154

132

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We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3Ј-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs Cold Spring Harbor Laboratory Press on May 10, 2018 -Published by genome.cshlp.org Downloaded from annotated in the FANTOM-2 annotation. We have also produced 547,149 5Ј end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, 5Ј-end clusters identify regions that are potential promoters for 8637 known genes and 5Ј-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.[Supplemental material available online at www.genome.org.]One of the primary goals of genome sequencing projects is to identify the genome sequences that are transcribed into functional mRNAs, so that full-length cDNAs can be isolated to allow further downstream biology, and functional and structural genomics. The limitations of a priori genome annotation dictate that the transcriptome needs to be identified experimentally via cDNA cloning and sequencing. Although expressed sequence tags (ESTs) (Adams et al. 1991(Adams et al. , 1995Hillier et al. 1996;Marra et al. 1999;Kargul et al. 2001) and ORESTES (Camargo et al. 2001) have been extremely valuable for new gene discovery, these approaches have not allowed highthroughput recovering of full-length cDNA clones nor definition of protein sequence derived from actual cDNA clones. To overcome such problems, we undertook from the year 1995, a strategic project aimed at the comprehensive collection of at least one full-length cDNA derived from each mouse gene, a strategy that is recently becoming useful in similar projects to collect full-length gene collections (Stapleton et al. 2002;Strausberg et al. 2002).Because of the limited processivity of reverse transcriptase and other limitations, standard cDNA libraries generally contain a majority of truncated transcripts. The introductio...

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“…These observations reveal that, while the current exon annotation of Xenopus laevis genome is far from complete, gene annotation is good enough for a high-performance junction-centric approach. Annotation of transcriptional units can rely on specific methodologies such as CAGE to map transcription start sites (Carninci et al, 1996) , or RNA-PET dedicated to the identification of gene boundaries (Peters and Velculescu, 2005). In addition, expressed sequence tags (EST) provide some hundreds of nucleotides of sequences originating from the 5' or 3' end of expressed mRNAs.Many X. laevis and tropicalis ESTs are represented in the databases (677911 and 1271480 in deEST release 130101,respectively),which probably contributes to the good quality of transcription unit annotation, hence the superior performances of the junction-centric approach.…”

Section: Discussionmentioning

confidence: 99%

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis

Noiret

Méreau

Angrand

et al. 2017

Developmental Biology

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Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exoncentric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed.We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

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High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper

Cited by 283 publications

References 2 publications

Cloning and Functional Analysis of cDNAs with Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

Cloning and Functional Analysis of cDNAs with Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells

Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis

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