Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1994-04-26
1998-09-15
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 5, 435 6, 435 912, 536 231, 536 243, C12Q 100, C12Q 170, C12Q 168, C07H 2102
Patent
active
058076697
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention deals with methods that allow the ultrasensitive detection of reverse transcriptase (RNA-dependent DNA-polymerase or deoxynucleoside triphosphate:DNA deoxynucleotidyl transferase) in a sample.
The invention includes both screening kits for the detection of reverse transcriptase by means of these methods and typing kits which relate reverse transcriptase activity to a certain agent or a group of agents with certain common features.
BACKGROUND OF THE INVENTION
Reverse transcriptase (referred to as RT in this text) is an indispensable component of mature virions of replication-competent retroviruses (Baltimore D. Nature 1970; 226:1209-1211; Temin HM and Mizutani S. Nature 1970; 226:1211-3). However, enzymes showing activites at least in part similar can also occur in a number of other retroelements, which do not belong to the family of retroviruses. On the one hand these include the retrovirus-like elements, which are subdivided into retrotransposons, pararetroviruses (caulimoviruses and hepadnaviruses) and LINE-elements, on the other hand the non-retrovirus-like elements such as mitochondrial plasmids or msDNA (for a recent survey of retroelements see Ricchetti M. Bull Inst Pasteur 1991; 89:147-58). Therefore this invention also describes methods for the ultrasensitive detection of all retroviruses as well as other retroelements which contain or express active RT. RTs are enzymes (RNA-dependent DNA polymerases) that, based on a given template RNA (substrate), produce the complementary DNA (cDNA). The result of this reaction, which is referred to as reverse transcription reaction (RT reaction), is a RNA/cDNA heteroduplex. A further activity of retroviral RTs is a RNase H function. Finally they also have DNA-dependent polymerase activity. For a survey on the properties of retroviral reverse transcriptase see H. Varmus and R. Swanstrom: Replication of Retroviruses; in: RNA Tumor Viruses, 2nd ed. R. Weiss, N. Teich, H. Varmus, J. Coffin, edts. Cold Spring Harbor Laboratory 1984 and 1985, chpts. 5 and S5.
All functional tests currently used for the detection of RT are based on the RNA-dependent synthesis of DNA. The natural template for the enzyme consists of the 70S genomic RNA contained in the virions. However, by using synthetic RNA homopolymers efficiency is increased 10 to 1000 fold. Independent of whether the template is homo- or heteropolymeric, the RT reaction depends of the presence of a suitable RT primer. Under natural circumstances this is a tRNA characteristic of the respective virus, though in most diagnostic RT assays oligodeoxyribonucleotides are used as primers. The most commonly used template-primer combinations today are homopolymers of the poly(rA)-oligo(dT) type. Since these can also be used by a number of eucaryotic DNA polymerases, they are frequently replaced by poly(rC)-oligo(dG). However, this combination can still be used by certain procaryotic DNA polymerases. These enzymes can even efficiently use heteropolymeric RNA templates (Gerard GF and Grandgenett DP. Retrovirus Reverse Transcriptase. In: Molecular Biology of RNA Tumor Viruses, JR Stephenson ed., Academic Press, New York, 1980, chpt. 9).
A standard procedure commonly used today for the detection of RT can be substrate to which a synthetic deoxyribonucleotide oligomer dT (or dG, resp.) of approximately 12 to 24 bases length is hybridized as a primer. After suitable preparation the sample containing the RT is added to a reaction mixture containing the template-primer combination, .sup.3 H-marked nucleoside triphosphate dTTP (resp. dGTP), a suitable divalent cation (Mg++or Mn++), and possibly other substances supporting the reaction, in a suitable buffer system. After incubation for a certain time and at a suitable temperature the reaction is stopped, the heteroduplex is precipitated by adding trichloric acid and transferred to a filter. The dried filter is then added to scintillation fluid, and the incorporated radioactivity is measured by means of liquid scintillation techniques (see U.S.
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Centers for Disease Control and Prevention, Federal Register, 59(200):52550 (Oct. 18, 1994).
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Boni Jurg
Schupbach Jorg
Jones W. Gary
Whisenaut Ethan
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