scholarly journals High-efficiency cloning of full-length cDNA.

1982 ◽  
Vol 2 (2) ◽  
pp. 161-170 ◽  
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Plasmid Dna ◽  
High Efficiency ◽  
Full Length ◽  
Mrna Sequence ◽  
Beta Globin ◽  
Cdna Synthesis ◽  
Globin Mrna ◽  
Full Length Cdna ◽  
Coding Regions ◽  
Entire Nucleotide Sequence

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

High-efficiency cloning of full-length cDNA

1982 ◽  
Vol 2 (2) ◽  
pp. 161-170
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Plasmid Dna ◽  
High Efficiency ◽  
Full Length ◽  
Mrna Sequence ◽  
Beta Globin ◽  
Cdna Synthesis ◽  
Globin Mrna ◽  
Full Length Cdna ◽  
Coding Regions ◽  
Entire Nucleotide Sequence

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

scholarly journals Aptamer-dependent full-length cDNA synthesis by overlap extension PCR

BioTechniques ◽  
2004 ◽  
Vol 37 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Yasumasa Mitani ◽  
Takayuki Nakayama ◽  
Matthias Harbers ◽  
Yoshihide Hayashizaki
Keyword(s):  
Full Length ◽  
Cdna Synthesis ◽  
Full Length Cdna ◽  
Overlap Extension Pcr ◽  
Overlap Extension

scholarly journals High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper

1998 ◽  
Vol 15 (5) ◽  
pp. 707-720 ◽  
Author(s):  
Motoaki Seki ◽  
Piero Carninci ◽  
Yoko Nishiyama ◽  
Yoshihide Hayashizaki ◽  
Kazuo Shinozaki
Keyword(s):  
High Efficiency ◽  
Full Length ◽  
Full Length Cdna

scholarly journals Identifying transcript 5′ capped ends in Plasmodium falciparum

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11983
Author(s):  
Philip J. Shaw ◽  
Jittima Piriyapongsa ◽  
Pavita Kaewprommal ◽  
Chayaphat Wongsombat ◽  
Chadapohn Chaosrikul ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Function ◽  
Malaria Parasite ◽  
Full Length ◽  
Human Malaria Parasite ◽  
Full Length Cdna ◽  
Coding Regions ◽  
Sequence Patterns ◽  
Mrna Transcripts ◽  
Generation Sequencing

Background The genome of the human malaria parasite Plasmodium falciparum is poorly annotated, in particular, the 5′ capped ends of its mRNA transcripts. New approaches are needed to fully catalog P. falciparum transcripts for understanding gene function and regulation in this organism. Methods We developed a transcriptomic method based on next-generation sequencing of complementary DNA (cDNA) enriched for full-length fragments using eIF4E, a 5′ cap-binding protein, and an unenriched control. DNA sequencing adapter was added after enrichment of full-length cDNA using two different ligation protocols. From the mapped sequence reads, enrichment scores were calculated for all transcribed nucleotides and used to calculate P-values of 5′ capped nucleotide enrichment. Sensitivity and accuracy were increased by combining P-values from replicate experiments. Data were obtained for P. falciparum ring, trophozoite and schizont stages of intra-erythrocytic development. Results 5′ capped nucleotide signals were mapped to 17,961 non-overlapping P. falciparum genomic intervals. Analysis of the dominant 5′ capped nucleotide in these genomic intervals revealed the presence of two groups with distinctive epigenetic features and sequence patterns. A total of 4,512 transcripts were annotated as 5′ capped based on the correspondence of 5′ end with 5′ capped nucleotide annotated from full-length cDNA data. Discussion The presence of two groups of 5′ capped nucleotides suggests that alternative mechanisms may exist for producing 5′ capped transcript ends in P. falciparum. The 5′ capped transcripts that are antisense, outside of, or partially overlapping coding regions may be important regulators of gene function in P. falciparum.

Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products

1992 ◽  
Vol 12 (3) ◽  
pp. 1149-1161
Author(s):  
S K Lim ◽  
C D Sigmund ◽  
K W Gross ◽  
L E Maquat
Keyword(s):  
Premature Termination ◽  
Degradation Products ◽  
Mrna Translation ◽  
Mrna Degradation ◽  
Full Length ◽  
Cytoplasmic Process ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Nonsense Codons

Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage.

scholarly journals Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

1992 ◽  
Vol 12 (3) ◽  
pp. 1149-1161 ◽  
Author(s):  
S K Lim ◽  
C D Sigmund ◽  
K W Gross ◽  
L E Maquat
Keyword(s):  
Premature Termination ◽  
Degradation Products ◽  
Mrna Translation ◽  
Mrna Degradation ◽  
Full Length ◽  
Cytoplasmic Process ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Nonsense Codons

Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage.

scholarly journals Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing

BioTechniques ◽  
2001 ◽  
Vol 31 (5) ◽  
pp. 1042-1049 ◽  
Author(s):  
Y. Shibata ◽  
P. Carninci ◽  
K. Sato ◽  
N. Hayatsu ◽  
T. Shiraki ◽  
...  
Keyword(s):  
High Efficiency ◽  
Full Length ◽  
Cdna Libraries ◽  
Full Length Cdna
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