Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1993-12-08
1999-02-16
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435912, C12Q 168, C12P 1934
Patent
active
058719083
DESCRIPTION:
BRIEF SUMMARY
This invention is directed to a process for determining at least one in vitro amplified nucleic acid in a reaction chamber, a device suitable for operating the process of the invention, and means for operating the process of the invention.
Increasingly, modern analytics returns to basic principles of molecular biology. Very recently, the polymerase chain reaction (PCR) was found to be a very useful tool for analytics utilizing basic principles of molecular biology, and is applicable to all the fields of analytics where nucleic acids play a direct or indirect role. al., Proc. Natl. Acad. Sci. 87, 1874-1878 (1990)! are able to detect low titers of DNA or RNA copies in aqueous solution. Ultimately, a single copy is sufficient for enzymatic amplification. However, a number of problems have shown in routine qualitative application as well as in the efforts to combine PCR with a quantitative copy number determination. The difficulties of amplification analytics are associated with: protein analytics.
WO 91/02815 describes the reliable qualitative and quantitative detection of specific DNA and RNA from biological sample material using a DNA/RNA amplification method in combination with, e.g., temperature gradient gel 6733-6734 (1990); J. Kang et al. Biotech. Forum Europe 8, 590-593, (1991); G. Gilliland et al. Proc. Natl. Acad. Sci. 87, 2725-2729 (1990)!.
By way of the amplification strategy described in WO 91/02815 it has been accomplished to combine the sensitivity of the amplification techniques with reliable and extremely precise quantification (variation .+-.15%). The method allows for controlling or overcoming the above-mentioned problems as are occurring with amplification processes. Using a nearly ideal internal standard of a defined number of copies, which standard differs from the template to be determined in only one single base position, amplification may be carried out with the initial ratio of Acids Res. 19, 6733-6734 (1990)!. Template and standard are made subsequently in a temperature gradient process using a time- or space/temperature gradient in the gel electrophoresis system. The ratio of template and standard may be determined in a simple fashion by hybridizing the reaction mixture with a radioactive-labeled or fluorescent-labeled standard. Heteroduplex formation, in other cases occurring interferingly, cannot distort the results in such system. Rather, heteroduplex formation is utilized for the actual measurement.
Usual problems caused by non-uniform consumption of primers, mispriming reactions, saturation effects, and variations in amplification efficiency do not have any negative influence on the results of quantification.
This technology has been applied to major analytical issues in medicine. Thus, for example, it has become possible to quantifyingly detect cytomegalic infections or viremia in transplanted patients or newborn children. Here, the process has proven particularly worthwhile since, in view of infection rates in the European countries of more than 90% here and there, the sole detection of cytomegalic viruses is of minor importance if not a titer determination is carried out simultaneously indicating the acute condition of viremia. With viral diseases, the quantifying aspect is gaining significance, particularly in context with the increasingly applied models of antiviral therapy. Frequently, antiviral therapies, as for instance in the case of HIV using AZT, are systemically extremely straining for the patient and, as an antiviral therapeutic agent, can only be employed for a limited period of time. In future, therefore, it will be very important to have an economic and simple technique allowing for both titer determination or determination of virus gene activity and indicating therapy-induced drift to resistant virus strains.
Having from small to low sample numbers (10-20), hitherto applied gel electrophoretic methods for separating the marked homoduplexes and heteroduplexes have proven extraordinarily efficient and correct with respect to the results supplied. However, a sever
REFERENCES:
patent: 5184020 (1993-02-01), Hearst et al.
Y. Jinno et al., "Use of psoralen as extinguisher of contaminated DNA in PCT", Nucleic Acids Research, Bd. 18, Nr. 22, p. 6739.
K. Henco et al., "Quantitative PCR: the determination of template copy numbers by temperature gradient gel electrohoresis (TGGE)", Nucleic Acids Research, Bd. 18, Nr. 22, pp. 6733-6734, 1990.
J.E. Hearst, "A photochemical investigation of the dynamics of the oligonucleotide hybridization", Ann. Rev. Phys. Chem., Bd. 39, pp. 291-315, 1988.
Kingsbury et al., "Rapid Detection and Identification of Infection Agents", Academic Press, Inc., pp. 245-256, 1985.
Eigen Manfred
Henco Karsten
Riesner Detlev
Evotec BioSystems GmbH
Horlick Kenneth R.
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