Reverse transcriptase purification

Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991). Rapid purification and char-aderisation of HIV-1 reverse transcriptase and RNase H engineered to incorporate a C-terminal tripeptide alpha-tubihn epitope, f EBBS Lett. 283,298-302. 

Roth, M. J., Tanese, N., and Goff, S. P. (1985). Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J. Biol. Chem. 260, 9326-9335. 

In the indirect labeling method, amine modified cDNA is first synthesised by incorporating aminoallyl-modified nucleotides in first strand cDNA by a reverse transcriptase. After hydrolysis of the RNA template, and purification of the amine-modified cDNA, chemical labeling with N-hydroxyl succinimidyl-ester derivative of the Cy dye is performed. A high excess of Cy dye NHS-ester is needed for an efficient reaction. The Cy dye-cDNA is then purified to remove Cy dye that is not incorporated into labeled cDNA. 

Vol. XXIX [15a]. Purification and Detection of Reverse Transcriptase in Viruses and Cells. D. L. Kacian and S. Spiegelman. 

Nakashima, H. Kudo, Y. Kobyashi, N. Motoki, Y. Neushul, M. Yamamoto, N. Purification and Characterization of an Avian Myeloblastosis and Human Immunodeficiency Virus Reverse Transcriptase Inhibitors, Sulfated Polysaccharides Extracted from Sea Mgdit Antimicrob. Agents Chemother. 1987, 31, 1524-1528. 

Nakashima H, Kido Y, Kobayashi N, Motoki Y, Neushul M, Yamamoto N (1987) Purifications and characterization of an avian myoblastosis and human immunodeficiency virus reverse transcriptase inhibitor sulfated polysaccharides extracted from sea algae. Antimicrob Agents Chemoth 31 1524-1528... 

Initially discovered in extracts of calf thymus as a contaminating enzyme during the purification of RNA polymerase (1,2), RNase H has since been found almost ubiquitously in lower and higher eukaryotes as well as in prokaryotes. A single cell type may have one or more species of RNases H residing in the cell nucleus and/or cytosol. Some RNases H have a single ribonucleolytic reaction specificity, whereas others are associated with DNA polymerase activities. Cellular RNases H, for example, from E. coli and calf thymus, are endonucleases the retroviral reverse transcriptase-associated RNases H can function as both exo- and endoribo-nucleases depending on the type of substrates available. 

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