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Cloning of noncultivatable human rotavirus by single primer amplification

Cloning of noncultivatable human rotavirus by single primer amplification
Cloning of noncultivatable human rotavirus by single primer amplification
A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.
0022-538X
1817-1822
Lambden, P.R.
e99ecc21-50d7-4a43-9e79-efba46592c77
Cooke, S.J.
01ed572d-e621-4c90-be53-97ac1b1bfdcd
Caul, E.O.
3ab60e6e-ca41-4196-a205-4c0075fd3767
Clarke, I.N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Lambden, P.R.
e99ecc21-50d7-4a43-9e79-efba46592c77
Cooke, S.J.
01ed572d-e621-4c90-be53-97ac1b1bfdcd
Caul, E.O.
3ab60e6e-ca41-4196-a205-4c0075fd3767
Clarke, I.N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b

Lambden, P.R., Cooke, S.J., Caul, E.O. and Clarke, I.N. (1992) Cloning of noncultivatable human rotavirus by single primer amplification. Journal of Virology, 66 (3), 1817-1822. (PMID:1371174)

Record type: Article

Abstract

A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.

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Published date: March 1992
Organisations: Faculty of Medicine

Identifiers

Local EPrints ID: 352643
URI: https://eprints.soton.ac.uk/id/eprint/352643
ISSN: 0022-538X
PURE UUID: 02c4b579-b519-4df5-ae06-e320cade7bdf
ORCID for I.N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 04 Jun 2013 12:51
Last modified: 06 Jun 2018 13:19

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Contributors

Author: P.R. Lambden
Author: S.J. Cooke
Author: E.O. Caul
Author: I.N. Clarke ORCID iD

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