Experiment Table CD4+ T lymphocyte counts and viral RNA Results Tetramer Staining Results Neutralizing Antibody Assays Results Viral Sequencing Results Peptide-binding Assays Results ELISPOT assays Results Conclusion   Navigation Home Methods Results Conclusion Quiz Links

Viral Sequencing Synopsis: The researchers sought to determine if there was a genetic alteration within certain genes of the viral genome that may have allowed the viral escape. The researchers used 500bp regions from the gag/pol/env region for sequence analysis. By sequencing these sample regions and carefully comparing the changes that occur over time the researchers hope to get an idea of the extent the entire genome is changing and perhaps identify the changes that enable the virus to escape from background mutations.   What factors might the researchers consider when picking genomic regions to compare? Answer They should pick regions that code for structural genes or for genes that may be involved in allowing viral escape.   Procedure: 1. These researchers analyzed 500 bp regions of gag/pol/env region of the genome from 8 to 12 viral clones per animal. 2. Blood samples were drawn and plasma was isolated by centrifugation. 3. Virions were lysed and RNA precipitated with Isopropanol, then solubilized. 4. Reverse Transcriptase was used for first strand synthesis using an SIV gag primer. 5. cDNA was produced and PCR amplification was performed. What is one of the main reasons for converting RNA to cDNA? Answer The devices that copy RNA are more error prone than DNA mechanisms. This is a liability in experiments where extensive manipulation of of genetic material takes place   6. These PCR products were spliced into plamsids (pCRII or pAMPI) and bacterial cells transformed.  7. These plasmids were isolated from the bacteria and were sequenced using T7/SP6 dideoxy sequencing.     What is one of the methods the researchers might use to pick out sucessfully transformed colonies? Answer There are several ways sucessfully transformed colonies could be picked out for example the plasmid might encode a cell phenotype. Also viral inserts could disrupt a gene in the plasmid. Here an absence of a phenotype would be searched for. NEXT