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.
Department of Microbiology, University of Southampton Medical School, Southampton General Hospital, United Kingdom.
P R
PR
Lambden
S J
SJ
Cooke
E O
EO
Caul
I N
IN
Clarke
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003967:Diarrhea / Q000382:microbiology; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D015678:Viral Structural Proteins; D012194:RNA-Directed DNA Polymerase