Fig. 1.

Construction of PRRSV NVSL #97-7895 full-length cDNA. (A) Five overlapping regions of the viral genome were amplified to generate the cDNA fragments A, B, 5C, 3C, and D. Fragment D was amplified to introduce 41 adenosines at the 3′ terminus. The T7 RNA polymerase promoter (Φ10) was inserted upstream of viral genomic 5′-terminal sequences in fragment A. Restriction enzyme sites used for cloning are listed below the fragments. (B) cDNA fragments with the consensus amino acid sequence were selected for assembly of the full-length clone in pBR322. An AclI site was incorporated immediately downstream of the poly(A) tail. The artificially introduced genetic marker, a BsrGI restriction enzyme site at position nt 1170, is shown. Bent arrow shows the position and direction of transcription by T7 RNA polymerase. (C) Original bacterial stock carrying pFL12 was propagated six times in E. coli DH5α. Plasmid DNA from passage 1 (P1) and 6 (P6) bacterial cultures was extracted, digested with MfeI, and resolved by electrophoresis on a 0.9% agarose gel along with a size marker (M). Expected sizes of the products of digestion are 1525, 1704, 1882, 1906, 2617, 4527, and 5034 bp. Gel image is inverted.