Use of polymerase chain reaction for efficient cloning of dsRNA segments of infectious bursal disease virus.
A method is described for efficient cloning of the large RNA segment of infectious bursal disease virus (IBDV) after reverse transcription and cDNA amplification. Complementary DNA segments of IBDV were prepared using reverse transcriptase and specific primers homologous to the conserved region at the 3' end of the IBDV sequence. The resulting cDNA segments were amplified using Taq DNA polymerase and a pair of specific primers. Three separate primer pairs were used for amplification, each yielding a cDNA fragment of the predicted size. The amplified products were directly used for cloning into a cloning vector, pCR1000. Three overlapping cDNA clones, containing the entire coding region of the large RNA segment of IBDV, were obtained. The identity of these clones was confirmed by hybridization with IBDV-specific probe, as well as by sequence analysis. By this method, approximately 3.2 kilobases of the large genome segments of two different strains of IBDV were cloned, and their complete nucleotide sequence was determined.
Vakharia
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VN
Center for Agricultural Biotechnology, Maryland Biotechnology Institute, University of Maryland, College Park 20742.
Ahamed
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B
He
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D007243:Infectious bursal disease virus / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*
D004277:DNA, Single-Stranded; D012330:RNA, Double-Stranded; D012367:RNA, Viral