Method of determining polymerase activity

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage

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435 6, 435 911, 435 772, 435 15, C12Q 168, C12Q 170, C12P 1934

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054139066

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BRIEF SUMMARY
The invention adresses a method of determining polymerase activity, a reagent and a reagent kit for implementing said method and the use of said method for the detection of HIV. The invention also addresses a method of determining the inhibitory effect of substances on polymerase activities, a method of detecting antibodies to polymerase and a method of detecting promoter sequences.
Polymerases are enzymes of fundamental importance to living beings. They are responsible, for example, for the synthesis of nucleic acids and their transformation into other nucleic acids necessary for the synthesis of proteins. Polymerases are, therefore, found in all types of cells including, for example, bacteria. Even many viruses code for their own polymerases. One special representative is a polymerase known as reverse transcriptase (RT).
With the discovery of human pathogenic retroviruses in the last decade (Science (1983), 220:868-871, Proc. Natl. Acad. Sci. USA (1980), 77:7415-7419 and Biochem. Biophys. Res. Comm. (1984), 121:126-133), the detection of the reverse transcriptase (RT), being the typical key enzyme of this virus family, has gained more and more importance. Numerous improvements of the method of determining RT-activity have since been described (Virology (1985) 147:326-335). RT-tests are commonly used in the following 3 situations: differentiate between different retroviruses, subject known to be infected with a retrovirus or a subject that may be infected and these enzymatic functions, that are essential for retroviruses.
With respect to the first two of the above mentioned fields of application, the known RT-test is limited in that it involves tedious sample preparation (ultracentrifugation or PEG precipitation, sensitivity may be too low otherwise); as regards the second field of application, the competition by sensitive, less expensive and simpler methods of antigen detection (ELISA); what limits all three of the above fields of application is the fact that conventional RT-tests use isotope nucleotides thus making special demands on the laboratory (equipment, authorization, personnel, waste disposal).
All methods of detecting enzyme activity of a reverse transcriptase, particularly reverse transcriptase of HIV, that are known from literature are based on assays, where a primer, which is complementary to a partial sequence of a template, is extended by incorporating labelled nucleotides. Non-incorporated nucleotides are usually separated from the extended primer that is bound to the template via hydrogen bonds by precipitating the polymer with the aid of trichloroacetic acid or ethanol followed by a filtration procedure or by adsorption of the polymer to a membrane filter having a positively charged surface (e.g. to DEAE, as described in Journal of Virological Methods 19 (1988), 161-168); or by employing any other method suitable to separate a negatively charged polymer from a negatively charged monomer. These measures are very tedious and require additional handling steps.
WO 90/06373 describes a method which uses immobilized nucleic acids as template nucleic acids. A drawback of this method is that sufficient specific immobilization of this template nucleic acid without obstructing the access for enzymes is not possible. The method can, hence, be limited by the available amount of reactive template nucleic acids. Further, the method produces a large number of different nucleic acids for which the binding to the solid phase via the template-DNA greatly varies as a consequence of different hybridisation conditions. Moreover, reactions at a solid phase generally exhibit poorer kinetics than do reactions in solutions.
Moreover, it has been found that for the quantitative determination of HIV-particles, immunoassays for the detection of HIV-antigens are not reliable (New England Journal of Medicine(1989). Vol. 321, 24, 1673-1675).
J. Clin. Mircobiology 27 (1989), 7, 1453-1455 and AIDS Research and Human Retroviruses 5 (1989), 535-540 disclose a method of detecting antibodies to HIV-RT which is based on the

REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
Advani et al. J. Clin. Microb. 27(7) 1453-1455 (1989).
Lee et al. J. Clin. Microb. 25(9) 1717-1721 (1987).
Day et al. Biochem. Journal 267:119-123 (1990).
Arias et al. J. Biol. Chem. 264(6):3223-3229 (1989).

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