Method for the detection of nucleic acids

Claim 2

Process according to claim 1, characterised in that one uses digoxigenin or digoxin as steroid.

Claim 3

Process according to claim 1 or 2, characterised in that the hapten is bound via the bridge to the C₅-position of uracil or cytosine or to the C₈-position of adenine or guanine.

Claim 4

Process according to claim 1 or 2, characterised in that the hapten is bound via the bridge to the 2'-position of the ribose.

Claim 5

Process according to one of claims 1 to 4, characterised in that a nucleic acid probe is used, the hapten of which has been incorporated into the nucleic acid probe enzymatically with the help of DNA polymerases, RNA polymerases, reverse transcriptases or terminal transferases and corresponding hapten-modified desoxy- or ribonucleoside triphosphate substrates.

Claim 6

Process according to one of claims 1 to 4, characterised in that a nucleic acid probe is used, the hapten of which is incorporated into the nucleic acid probe photochemically with the help of photohapten.

Claim 7

Process according to one of claims 1 to 4, characterised in that a nucleic acid probe is used, the hapten of which has been incorporated into the nucleic acid probe chemically in the course of the oligodesoxyribonucleotide synthesis by incorporation of protected nucleoside phosphoamidites modified with substitutable amino functions and - after removal of the protective groups - by reaction of the modified oligo-desoxyribonucleotide with activated esters, amides or ethers of the hapten.

Claim 8

Process according to one of claims 1 to 7, characterised in that, as labelling of the anti-hapten antibody, there is used an enzyme labelling, radioactive labelling, fluorescence labelling or (bio)luminescence labelling.

Claim 9

Process according to one of the preceding claims, characterised in that the nucleic acids to be detected are detected by in situ hybridisation in cells fixed on microscope slides.

Claim 10

Process according to one of claims 1 to 8, characterised in that the nucleic acids to be detected are detected by in situ hybridisation in tissue smears.

Claim 11

Process for the detection of nucleic acids, characterised in that, after denaturing, the nucleic acid to be detected (a) is brought to hybridisation with at least two primers of which a first one is, in its sequence, complementary to a part sequence of a strand of the nucleic acid to be detected and a further one is identical with another part sequence of the same strand of the nucleic acid to be detected, (b) is treated with polymerase, desoxyribonucleotides and at least one desoxyribonucleotide, which, as hapten, contains a steroid chemically bound, which, on at least one position of the desoxyribonucleotide which is not involved in the hydrogen bridge formation, is bound via a bridge of at least 4 atoms length, (c) the cycle consisting of denaturing, hybridisation and polymerisation is repeated at least once and (d) the resultant labelled nucleic acid is detected via an antihapten antibody which is itself labelled.

Claim 12

Process for the detection of nucleic acids, characterised in that, after denaturing, the nucleic acid to be detected (a) is brought to hybridisation with at least two primers of which a first one is, in its sequence, complementary to a part sequence of a strand of the nucleic acid to be detected and a further one is identical with another part sequence of the same strand, (b) is treated with desoxyribonucleotides and a polymerase, (c) subsequently, the cycle consisting of denaturing, hybridisation and polymerisation is repeated at least once, (d) subsequently, a cycle is carried out in the presence of at least one desoxyribonucleotide, which, as hapten, contains a steroid chemically bound which, on at least one position of the desoxyribonucleotide which is not involved in the hydrogen bridge formation, is bound via a bridge of at least 4 atoms length and (e) the resulting labelled nucleic acid is detected via an antihapten antibody which is itself labelled.

Claim 13

Process according to claim 11 or 12, characterised in that, after completion of the cycles, a nucleic acid immobilised on a solid phase, complementary to the nucleic acid to be detected, is added, a hybridisation is carried out and, after separation of solid and liquid phase, the labelling is determined in the solid phase.

Claim 14

Process according to one of claims 11 to 13, characterised in that one uses the Taq-DNA polymerase as polymerase.

Claim 15

Derivatised nucleic acid which, via a chemical bond, contains at least one hapten as labelling, characterised in that the hapten is a steroid which, on at least one position of the nucleic acid which is not involved in the hydrogen bridge formation, is bound via a bridge of at least 4 atoms length.

Claim 16

Nucleic acid according to claim 15, characterised in that the steroid is digoxigenin.

Claim 17

Use of a nucleic acid according to claim 15 as probe for the detection of complementary nucleic acids.

Claim 18

Desoxyribo- and ribonucleoside triphosphate which, as labelling, contains a chemically bound hapten, characterised in that the hapten is a steroid which is bound via a bridge of at least 4 atoms length to a position which is not involved in the hydrogen bridge formation.

Claim 19

Desoxyribo- and ribonucleoside triphosphate according to claim 18, characterised in that the steroid is digoxigenin.

Claim 20

Process for the production of a derivatised nucleic acid according to claim 15, whereby the hapten is incorporated enzymatically with the help of DNA polymerases, RNA polymerases, reverse transcriptases or terminal transferases and a desoxyribo- or ribonucleoside triphosphate according to claim 18.

Claim 21

Process for the production of a derivatised nucleic acid according to claim 15, whereby, in the course of the oligodesoxyribonucleotide synthesis according to the phosphite triester method, besides the protected nucleoside phosphoramidites, protected nucleoside phosphoramidites modified with substitutable amino functions are precisely incorporated into the oligodesoxyribonucleotide single strand, the protective groups are removed and the resultant single-stranded oligodesoxyribonucleotide is reacted with an activated ester, amide or ether of the steroid.