A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes.
Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.
Zhou
Fuchun
F
Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229, USA.
Li
Qiuhua
Q
Gao
Shou-Jiang
SJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004267:DNA Viruses / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry; D005814:Genes, Viral; D016679:Genome, Viral; D018144:Rhadinovirus / Q000235:genetics / Q000254:growth & development
D004251:DNA Transposable Elements; D004279:DNA, Viral