Outline of the SISPA method. A. Viral RNA is converted to cDNA using random-tagged and poly-A tagged primers (FR26RV-N and FR40RV-T). B. Second strand DNA is synthesized using Klenow exo-DNA polymerase, in the presence of random tagged and virus specific 5' end oligo primers. C. Double stranded DNA is amplified by PCR using the primer tag (FR20RV). D. Amplicons are separated by electrophoresis and products ranging from 500–1000 nucleotides are cloned into the TOPO vector. 96–288 colonies are picked, plasmid DNA is purified and the inserts are sequenced.
Djikeng et al. BMC Genomics 2008 9:5 doi:10.1186/1471-2164-9-5