Full-Length cDNA Synthesis for Long-Distance RT-PCR of Large mRNA Transcripts

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.

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An assay for full-length cDNA synthesis from a heterogeneous population of mRNA, exemplified by opsin and the retina

Biochemical Society Transactions ◽

10.1042/bst0170516 ◽

1989 ◽

Vol 17 (3) ◽

pp. 516-518

Author(s):

CAROLYN L. DENT ◽

B. EDWARD H. MADEN

Keyword(s):

Heterogeneous Population ◽

Full Length ◽

Cdna Synthesis ◽

Full Length Cdna

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[41] Full-Length cDNA clones: Vector-primed cDNA synthesis

Methods in Enzymology - Guide to Molecular Cloning Techniques ◽

10.1016/0076-6879(87)52044-4 ◽

1987 ◽

pp. 371-389 ◽

Cited By ~ 8

Author(s):

Prescott L. Deininger

Keyword(s):

Full Length ◽

Cdna Clones ◽

Cdna Synthesis ◽

Full Length Cdna

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Identifying transcript 5′ capped ends in Plasmodium falciparum

PeerJ ◽

10.7717/peerj.11983 ◽

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.

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Development of Infectious cDNA Clones of Citrus Yellow Vein Clearing Virus Using a Novel and Rapid Strategy

Phytopathology ◽

10.1094/phyto-02-18-0029-r ◽

2018 ◽

Vol 108 (10) ◽

pp. 1212-1218 ◽

Cited By ~ 3

Author(s):

Tiantian Cui ◽

Yu Bin ◽

Jianhong Yan ◽

Pengying Mei ◽

Zhongan Li ◽

...

Keyword(s):

Causal Agent ◽

Full Length ◽

Yellow Vein ◽

Wild Type Virus ◽

Economic Losses ◽

Citrus Limon ◽

Cdna Clones ◽

Rt Pcr ◽

Long Distance ◽

Vein Clearing

Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch’s postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus−host interactions.

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High-efficiency cloning of full-length cDNA.

Molecular and Cellular Biology ◽

10.1128/mcb.2.2.161 ◽

1982 ◽

Vol 2 (2) ◽

pp. 161-170 ◽

Cited By ~ 833

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.

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Genome Structure and Production of Biologically Active In Vitro Transcripts of Cucurbit-Infecting Zucchini green mottle mosaic virus

Phytopathology ◽

10.1094/phyto.2002.92.2.156 ◽

2002 ◽

Vol 92 (2) ◽

pp. 156-163 ◽

Cited By ~ 26

Author(s):

Ju Yeon Yoon ◽

Byoung Eun Min ◽

Jang Kyung Choi ◽

Ki Hyun Ryu

Keyword(s):

Mosaic Virus ◽

Full Length ◽

Biologically Active ◽

Wild Type Virus ◽

Progeny Virus ◽

Close Relative ◽

Rt Pcr ◽

Free System ◽

Full Length Cdna

The complete nucleotide sequence of the Zucchini green mottle mosaic virus (ZGMMV), a new member of the genus Tobamovirus, has been determined. The genome of ZGMMV is 6,513 nucleotides long and contains four open reading frames coding for proteins of 131, 189, 28, and 17 kDa from the 5′ to 3′ end, respectively. The 5′- and 3′-non-translated regions consist of 59 and 163 residues, respectively. The sequences of the viral proteins exhibit high identity to the proteins of the members of the genus Tobamovirus and are distinct from other viruses within the subgroup of cucurbit-infecting tobamoviruses. Results from phylogenetic trees of the coding regions demonstrated that ZGMMV is a very close relative of Kyuri green mottle mosaic virus and Cucumber fruit mottle mosaic virus and is less similar to Cucumber green mottle mosaic virus. Full-length cDNA of ZGMMV was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using the 5′-end primer containing a T7 RNA promoter sequence and 3′-end primer. Capped in vitro transcript from the RT-PCR products was infectious on zucchini squash, cucumber, and Nicotiana benthamiana plants. This cell-free system to produce infectious transcripts from uncloned cDNA copies is useful for quick assessment of infectivity of transcripts from plant RNA viruses prior to cloning. Synthesized capped transcript from a full-length cDNA clone of the virus was highly infectious. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first report of a biologically active transcript from a cucurbit-infecting tobamovirus.

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