Reverse transcriptase method

Tonini, T., Claudio, P.P., Giordano, A., Romano, G. (2004). Retroviral and lentiviral vector titration by the analysis of the activity of viral reverse transcriptase. Methods Mol. Biol., 285, 155-158. 

Balzarini, J. and De Clercq, E. (1996) Analysis of inhibition of retroviral reverse transcriptase. Methods Enzymol. 275,472-502. 

There are two different methods to identify modified residues and ribonuclease scissions in RNA molecules the reverse transcriptase method or the end-labelling method. The choice of method depends both on the length of the studied RNA and the method of probing (as discussed in Sections 4.4.1 and 4.4.2). The reverse transcription method uses extension of a primer and therefore is independent of the length of the RNA, while the end-labelling method is restricted to analysis of small RNA molecules (n < 300). The latter method requires scission of the RNA. 

Modified residues can be detected by reverse transcriptase methods or end-labelled methods after an acidic aniline cleavage (Section 4.4.3). Hydrazine modifications can only be performed under denaturing conditions. 

CMCT Probing U (N3) > G (Nl). (Section 4.4.2.5, native conditions or follow Section 4.4.2.4, denaturing conditions), but incubate for 5 min at 95°C. Modified residues can be detected directly by reverse transcriptase methods. 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

RH Smith Jr, WL Jorgensen, J Tirado-Rives, ML Lamb, PAJ Janssen, CJ Michejda, MBK Smith. Prediction of binding affinities for TIBO inhibitors of HIV-1 reverse transcriptase using Monte Carlo simulations m a linear response method. J Med Chem 41 5272-5286, 1998. 

This acronym stands for Reverse Transcriptase Polymerase Chain Reaction, a method used to first copy a strand of RNA into cDNA, then amplify it through standard PCR methods. 

Hertogs K, de Bethune MP, Miller V, Ivens T, Schel P, Van Cauwenberge A, Van Den Eynde C, Van Gerwen V, Azijn H, Van Houtte M, Peelers F, Staszewski S, Conant M, Bloor S, Kemp S, Larder B, Pauwels R (1998) A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob Agents Chemother 42 269-276... 

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

Efavirenz (1) was chosen over compound 2 as a developmental candidate in 1993 based on its better antivirus activities, especially against resistant strains [1, 17]. Efavirenz is the first HIV non-nucleoside reverse transcriptase inhibitor (NNRTI) which was approved by the FDA on September 21, 1998. The original Medicinal Chemistry method to prepare Efavirenz is depicted in Scheme 1.14. 

The viral load test quantifies viremia by measuring the amount of viral RNA. There are several methods used for determining the amount of HIV RNA reverse transcriptase-coupled polymerase chain reaction, branched DNA, and nucleic acid sequence-based assay. Each assay has its own lower limit of sensitivity, and results can vary from one assay method to the other therefore, it is recommended that the same assay method be used consistently within patients. 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

An extension of the PCR method is RT-PCR. Here mRNA is extracted from the cells or tissue, converted to cDNA using the enzyme reverse transcriptase and PCR is then carried out This method is used to detect the expression of specific mRNA sequences in cells or tissues. 

Genotypic resistance assays use DNA sequencing methods to examine the reverse transcriptase and protease regions of fhe HIV genome for all resistance-associated mutations. A major drawback of fhis fesfing mefhod is that results are difficult to interpret and expert consultation is necessary. 

Aubry, A.-E. et al.. Column selection and method development for the determination of the enantiomeric purity of investigational non-nucleoside reverse transcriptase inhibitors. Chirality, 13, 193, 2001. 

Genetic methods Polymerase chain reaction (PCR) and its variants, e.g., multiplex PCR, are widely used for C. botulinum detection. Reverse-transcriptase PCR is recommended as an alternative to biological tests using mice (McGrath et al., 2000). 

Gussio, R., Pattabieaman, N., Kellogg, G.E., Zahaeevitz, D.W. Use of 3D QSAR methodology for data mining the National Cancer Institute Repository of Small Molecules application to HlV-1 reverse transcriptase inhibition. Methods 1998, 14, 255-263. 

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