Cloning of noncultivatable human rotavirus by single primer amplification

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.

Candidate new rotavirus species in Schreiber's bats, Serbia

“…To obtain the true sequence of the genome segment ends, a short oligonucleotide (PC3-mod), phosphorylated at the 5′ end and blocked at the 3′ end with dideoxy cytosine, was ligated to the 3′ ends of the genomic RNA in the nucleic acid extract (Lambden et al, 1992;Potgieter et al, 2002). In brief, 5 μL total RNA was combined with 25 μL RNA ligation mixture (consisting of 3.5 μL nuclease free water, 2 μL of 20 μM PC3, 12.5 μL of 34% (w/v) polyethylene glycol 8000, 3 μL of 10 mM ATP, 3 μL 10× T4 RNA Ligase buffer and 10 U T4 RNA Ligase I (New England Biolabs) and then incubated at 17°C for 16 h. Following the incubation, the RNA was extracted using the QIAquick Gel Extraction Kit (QIAGEN).…”

Metagenomics and the molecular identification of novel viruses

“…A sequence-independent amplification technique termed sequence-independent single primer amplification (SISPA) was introduced almost two decades ago to identify viral nucleic acid of unknown sequence present in low amounts . SISPA was used first to sequence the norovirus genome from human faeces (Matsui et al, 1991), in addition to an astrovirus (Matsui et al, 1993) and a rotavirus (Lambden et al, 1992) infecting humans. Originally the SISPA method involved endonuclease digestion of DNA, followed by directional ligation of an asymmetric adaptor or primer on to both ends of the DNA molecule .…”

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

“…AmCPV S2 RNA was converted to cDNA following a sequence independent RT method (Lambden et al, 1992) using two primers AG1 and AG2 as discussed by Chavali et al (2008). cDNA was then cloned into pCR-XL-TOPO vector to make plasmid pCR-XL-TOPO/ AmCPV S2.…”