Reverse transcriptase assay

The basis of a reverse transcriptase assay is to extend a deoxynucle-otide primer, annealed to the RNA, by reverse transcriptase. 

Modified bases in the RNA will stop or pause the reverse transcriptase 3 to the modified base. Breaks in the RNA induced by ribonucleases or chemicals will also terminate the polymerase. The electrophoretic analysis of the reverse transcript reveals the stops as bands on an autoradiograph. The position of the modification or scission can be determined by co-electrophoresis of a dideoxy sequence reaction, and the intensity of the band will reflect the reactivity of the base (Figs 4.7 and 4.10). 

The identification of the modified residue is established when the sample is co-electrophoresed with a sequence (Section 4.4.3.1.3). Since the reverse transcriptase stops or pauses 3 of the modified band, the dideoxy sequencing bands are displaced by one base relative to the modified base, i.e. if a residue is modified the corresponding band will appear as one nucleotide shorter than the base will appear in the sequence lane. 

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Prepare radioactive reverse transcriptase assay solution by adding [a-32P]-TTP to a final concentration of 20 pCi/mL of RT cocktail. Mix well and distribute 25 pL of this mixture/well into a U- or V-bottomed 96-well plate. 

Reverse Transcriptase Assay Based on Product Enhancement for Assessing the Drug Susceptibility of Retroviruses... 

Fig. 1. Schematic representation of the described ultrasensitive reverse transcriptase assay, using PCR for product enhancement.

Pyra, H., Boni, J., and Schupbach, J. (1994) Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc. Natl. Acad. Sci. USA 91, 1544-1548. 

Boni, J., Pyra, H., and Schupbach, J. (1996) Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1 infected individuals by the product-enhanced reverse transcriptase assay. J. Med. Virol. 49,23-28. 

Berberine chloride was evaluated in the human immunodeficiency virus reverse transcriptase assay and found to be moderately active (50 pg/ml < IC50 < 150 pg/ml)[229]. 

The modification at N1 positions on adenosines and N3 positions on cytidines is directly monitored by the reverse transcriptase assay. However, it is important to keep in mind that conditions which give rise to a limited level of modification of adenosines and cytidines may, in addition, substantially modify guanosines at the N7 position, which may lead to secondary effects. The extent of guano-sine modifications cannot be detected directly with the reverse transcriptase assay but can be assessed after reduction with sodium borohydride followed by acid-catalysed strand scission (Section 4.4.2.2). 

DEP-modified adenosines are monitored directly in reverse transcriptase assays. The reason for the termination of the reverse transcription is not clear. It may either reflect the bulkiness of the carbethoxy group on the N7, or that the carbethoxylation of N7 causes spontaneous hydrolysis at the C8 position and therefore breakage of the imidazole ring. A third explanation is that subsequent to the N7 modification the N6 position is carbethoxylated and this modification blocks the reverse transcriptase. 

Kethoxal is a 1,2-dicarbonyl compound that reacts with both the N1 and N2 positions on guanosines and generate a tricyclic adduct. Kethoxal reacts with guanosines in single-stranded regions and terminal base pairs. The modified residues are monitored directly in the reverse transcriptase assay. 

Detailed overviews on test methods and vimses detected are provided. For retro-vimses and other endogenous vimses, electron microscopy, infectivity assays or reverse transcriptase assays are proposed. Non-endogenous or adventitious vimses may be detected by suitable in vitro or in vivo assays or (in case of rodent cell fines) by a corresponding antibody production test in the respective species. Other virus-specific tests might be useful alternatives. 

Garciosaphenone A (38) showed interesting anti-HIV-1 activity [34], It was very active in HIV-1 reverse transcriptase assay (IC50 = 23.9 pg mL 1). Its activity was comparable to those of fagaronine hydrochloride, but 7-13 times less sensitive than nevirapine. Nevertheless, garciosaphenone A was toxic in the syncytium test. 

Triton X-100), and the lysate (40 pi) was subjected as an enzyme solution to an HIV-1 RT activity assay using a Reverse Transcriptase Assay nonradioactive kit (Roche Diagnostics Corp., Tokyo, Japan) in accordance with the manufacturer s instructions. 

HBV Liver cirrhosis Reverse transcriptase assay, interferons RT- PCR (HepG2 2.2.15)... 

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