Process for the detection of nucleic acids

Details Process for the detection of nucleic acids Process for the detection of nucleic acids

1990-01-09

1994-09-06

Schwartz, Richard A.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 243, 536 2532, C12Q 168

Patent

active

053447573

DESCRIPTION:

BRIEF SUMMARY
The invention concerns a process for the detection of nucleic acids of defined sequence by hybridization with complementary, labelled nucleic acid probe.
One of the most used molecular-biological techniques is the DNA/DNA, RNA/RNA or RNA/DNA hybridization for the detection of homologous nucleic acid sequences. A nucleic acid (DNA or RNA) probe is labelled and brought into contact with a nucleic acid (DNA or RNA) to be investigated, usually fixed on a filter, under hybridization conditions. If there is homology between the nucleic acids used as probe and the nucleic acid to be detected, these forms a hybrid double strand. The hybrids are subsequently detected. Hitherto, the labelling of the probe generally took place by incorporation of radioactively derivatized desoxyribonucleoside triphosphates therein. The detection of the hybrids then took place by autoradiography. Such conventional, radioactively-labelled DNA probes are very effective and sensitive but problems arise due to the need to handle radioactive material. Handling with radioactive material requires specially trained personnel since handling by unskilled individuals, leads to endangering of the laboratory safety. Furthermore, the disposal of radioactive materials is a further problem. In addition, the radioactively-labelled samples, because of the half-life times of the radioactive materials used, can only be used for a certain period of time after their preparation. When the detection of small amounts of DNA are to be detected, the necessary time of exposure of the autoradiography can also be very long, on line of days to weeks.
Besides the radioactively-labelled systems for the detection of nucleic acids, non-radioactive methods are known, whereby the nucleic acid samples used are modified with biotin molecules (U.S. Pat. No. 4,687,732, EP-A 0063879), digoxin-/T.sub.3 -/T4 molecules (EP-A-0173251) or with alkyl-/butyl-/ethyl-/sulphonic acid-
itroso molecules (EP-A 128018). The incorporation of these low molecular weight molecules into the complementary nucleic acid probe thereby takes place chemically, photochemically or enzymatically. They are then hydridized with the nucleic acid sequence to be detected. The detection of the hybrids then takes place via binding of the low molecular weight molecule by a (strept)avidin-labelling enzyme conjugate in the case of biotin, antidigoxin/-T3-/-T4-antibody-labelling enzyme conjugate in the the case of digoxin-/T3-/T4-molecules or via anti-alkyl-/-butyl-/-ethyl-/-sulphonic acid-/-nitroso-antibody-labelling enzyme conjugates. The detection of the hybridization product takes place by the determination of the enzymatic activity of the labelling enzyme, using coupled coloured materials systems. However, in the case of the method of EP-A-0 173 251, the binding of the digoxin-/T3-/T4 molecules takes place on an N-atom participating in the hydrogen bridge formation of one or more bases of the nucleic acid probe.
In these cases, hybridization is impaired, especially when the probe has been multiply modified. With the exception of the biotin/(strept)avidin system, the sensitivity of the known non-radioactive systems as compared to radioactive systems, is at least 10 to 100 times lower. The uniquely high sensitivity of the non-radioactive detection of biotin/(strept)avidin system is to be attributed to the high binding constant (K=10.sup.15 mol.sup.-1) See, e.g. Kinow; Proc. Natl. Acad. Sci. USA 80 (1983) 4045). The maximum achievable sensitivity of the biotin/(strept)avidin system lies, as also in the case of radioactive labelling, in the detection of from 0.1 pg. to 1 pg. DNA in the dot-blot assays and in detection of "single-copy" genes, i.e. of genes which occur only once the genome, in using from 1 to 10 .mu.g. of genomic DNA fragments in genomic blots. However, the utilization of the biotin/(strept)avidin system has a decisive disadvantage in that it is very subject to disturbance since the vitamin biotin occurs in almost all biological materials (Biochem. Biophys. Acta 29 (1985) 225; Biochem. Biophys. Ac

REFERENCES:
patent: 5198537 (1993-03-01), Huber et al.
Langer et al. (Nov. 1981), Proc. Natl. Acad. Sci. USA 78(11):6633-6637.
Hunter et al. (Sep. 1982), J. Immunol. 129(3):1165-1172.
Lewis et al. (1987), J. Clin. Pathol. 40:163-166.
Forster et al. (1985), Nucl. Acids. Res. 13(3):745-761.
Saiki et al. (1985), Science 230:1350-1354.

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