Reverse transcriptase reaction specificity

Mature mRNA transcripts (sense strand) from eukaryotic cells can be purified and then reverse transcribed, with the assistance of a reverse transcriptase enzyme (from Moloney murine leukemia virus, MMLV), into complementary DNAs (cDNAs) that will anneal with the mRNA transcripts by Watson-Crick base pairing to give anti-parallel DNA/RNA duplexes or double helices. The poly(A) tail in each mature mRNA transcript is actually a usefiil handle for each reverse transcriptase reaction. Thereafter, DNA/RNA duplexes must be broken down with the assistance ofRNAse enzymes (specific for the hydrolysis of RNA phospho diester links) and a sense strand of DNA constructed instead on each cDNA single strand so that equivalent, more stable antiparallel DNA/DNA duplexes are generated instead, with the assistance of a DNA polymerase enzyme. In this instance, the poly(T) tail in each cDNA molecule turns out to be important for the DNA polymerase reaction ... 

In the realm of natural product synthesis, Kepler and Rehder utilized the K-R reaction to synthesize ( )-calanolide A (56), a potent non-nucleosidal human irmnunodeficiency virus (HIV-1) specific reverse transcriptase inhibitor. Propiophenone 57 was allowed to react with acetic anhydride in the presence of sodium acetate to afford benzopyranone 58 in 56% yield subsequent deacetylation of 58 gave 59. Flavone 59 was then transformed to ( ) calanolide A (56) over several steps. 

Mangham DC, Williams A, McMullan DJ, et al. Ewing s sarcoma of bone the detection of specific transcripts in a large, consecutive series of formalin-fixed, decalcified, paraffin-embedded tissue samples using the reverse transcriptase -polymerase chain reaction. Histopathology 2006 48 363-376. 

A set of sense and antisense primers should be selected to synthesize specific cDNAs and also to detect the amplified messages of the genes, complementary to their specific gene sequences. It is important to consider the following points while designing the primers for reverse transcriptase and polymerase chain reaction ... 

Galvan, B. Christopoulos, T. K. Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA. Clin. Biochem. 1997, 30(5), 391-397. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Slawin KM, Shariat SF, Nguyen C, et al. Detection of metastatic prostate cancer using a splice variant-specific reverse transcriptase-polymerase chain reaction assay for human glandular kallikrein. Cancer Res 2000 60 7142-7148. 

Figure 13 Dose- and time-dependent induction of CYP3A4 (A), CYP3A5 (B), and CYP3A7 (C) mRNAs in human cultured hepatocytes incubated with rifampicin. Cells cultured on Matrigel-coated 96-well plates were incubated with increasing doses of rifampicin, and RNA was harvested at times indicated. Specific CYP mRNAs were determined by TaqMan RT-PCR. Abbreviations. CYP, cytochrome P450 RT-PCR, realtime reverse transcriptase-polymerase chain reaction.

Although a number of mammalian retrotransposons have been discovered by chance, a variety of approaches have been developed specifically to survey the genome for the presence of retrotransposons as well as to facilitate their isolation and characterization. Of the three methods outlined here in detail, two (conserved restriction sites and phylogenetic screening) could lead to isolation of all three types of retrotransposons, whereas the third method [polymerase chain reaction (PCR) amplification of reverse transcriptase] will exclude the SINEs. An unrelated method that has been particularly useful for isolation of SINEs7 will not be detailed. 

Dideoxynucleosides show potent anti-retroviral activity against HIV-specific reverse transcriptase80-83. In particular, 2, 3 -dideoxy-3 -C-cyano-2 -substituted thymidine derivatives (33 A and 33 B) with a free 5 -hydroxy function (R1 = H) are potential inhibitors of the HIV-reverse transcriptase-promoted c-DNA synthesis. As these compounds have yet to be prepared by another method, the 3 -ene-nitrile 3284 was subjected to conjugate addition reactions with ammonia, primary amines, secondary amines and carbon nucleophiles. Most of these nucleophilic amine addition reactions give either the trans-isomer 33 A as the sole product (e.g., reaction with pyrrolidine, piperidine, morpholine), or as the major product along with the c/s-isomer (e.g., reaction with methylamine, benzylamine), except for the reaction with ammonia where the cts-isomer 33B is formed as the major product84. 

The two platelet membrane glycoproteins for which the strongest evidence exists at the present time that they are thrombin receptors are a specific form of the GPIb-DC-V conq>lex and PARI, the protease-activated thrombin receptor these will be discussed next in this review. Based on their apparent ability to form complexes with a hrombin, several other proteins had previously t en proposed as thrombin receptors but in only a few cases were the necessary further studies carried out to test these hypotheses. Protease nexin 1 was proposed as a thrombin receptor based on the similar time courses of complex formation and platelet activation but it was subsequently found that protease nexin 1 cannot be a receptor since it is an internal conq>onent of platelets that is expressed on the surface only ater activation. Glycoprotein V has also been proposed as a thrombin receptor based on the feet that it can be cleaved by low concentrations of a-thrombin but subsequent studies (reviewed in ) showed that there was no consistent relationship between the rate or extent of GPV hydrolysis and the extent of platelet activation induced by a-thrombin. Another thrombin-activatable receptor, PAW, has been identified in mouse platelets but not in human platelets using a reverse transcriptase/ polymerase chain reaction approach (unpublished data and S.Coughlin, personal communication). PAR3 has, however, been reported cloned from a human platelet cDNA library. ... 

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