# US 5,344,757 A · Process for the detection of nucleic acids

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- Number: US 5,344,757 A
- Document ID: US5344757A
- Status: Granted
- Kind: A
- Application number: 07/415,307
- Filed: 1990-01-09
- Granted: 1994-09-06
- Earliest claimed: 1988-01-12
- Assignees: Boehringer Mannheim GmbH
- Inventors: Hans-Joachim Holtke; Rudolf Seibl; Gudrun Schmitz; Hans R. Scholer; Christoph Kessler; Ralf Mattes
- Web page: https://patentgrep.com/patent/US5344757A

## CPC

- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

For the detection of nucleic acids of definite sequence by hybridisation with a complementary nucleic acid probe which contains bound via a chemical bonding at least one hapten as labelling one uses, as hapten, a steroid which is bound via a bridge of at least 4 atoms length to at least one position of the nucleic acid probe which does not participate in hydrogen bridge formation and detects the hybridised probe via an in turn labelled anti-hapten antibody.

## Claims

### Claim 1

Method for detection of a first nucleic acid sequence comprising contacting said sequence with a complementary nucleic acid probe under conditions favoring hybridization between said sequence and said probe, wherein said probe is a second nucleic acid sequence having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said second nucleic acid sequence via a bridge of at least 4 atoms length at a position thereon which does not participate in formation of hydrogen bonds with said first sequence, contacting hybridized probe with a labelled antibody which specifically binds to said steroid hapten, and detecting said labelled antibody to detect said first nucleic acid sequence.

### Claim 2 (depends on 1)

Method of claim 1 wherein said bridge is from 4 to 32 atoms in length.

### Claim 3 (depends on 1)

Method of claim 1 wherein said bridge is from 11 to 16 atoms in length.

### Claim 4 (depends on 1)

Method of claim 1 wherein said bridge contains hydrophilic groups.

### Claim 5 (depends on 1)

Method of claim 1 wherein said bridge is linear.

### Claim 6 (depends on 1)

Method of claim 1 wherein said bridge is branched and contains a hapten molecule at at least one chain end.

### Claim 7 (depends on 1)

Method of claim 1 wherein said steroid hapten is bound to a nucleotide base via said bridge.

### Claim 8 (depends on 7)

Method of claim 7 wherein said base is uracil or cytosine and said steroid hapten is bound to C5 via said bridge.

### Claim 9 (depends on 7)

Method of claim 7 wherein said base is adenine or quanine and said steroid hapten is bound to C8 via said bridge.

### Claim 10 (depends on 1)

Method of claim 1 wherein said steroid hapten is bound to a ribose via said bridge.

### Claim 11 (depends on 10)

Method of claim 10 wherein said steroid hapten is bound to the 2' position of ribose via said bridge.

### Claim 12 (depends on 1)

Method of claim 1 wherein said steroid hapten is bound to said bridge via an ester, amide, or ether bond.

### Claim 13 (depends on 1)

Method of claim 1 wherein said antibody is labelled with an enzyme label, a radioactive label, a fluorescence label or a bioluminiscent label.

### Claim 14 (depends on 13)

Method of claim 13 wherein said antibody is labelled with an enzyme.

### Claim 15 (depends on 14)

Method of claim 14 wherein said enzyme is alkaline phosphatase, peroxidase or beta galactosidase.

### Claim 16 (depends on 13)

Method of claim 13 comprising detecting said labelled antibody via a redox reaction system.

### Claim 17 (depends on 16)

Method of claim 16 wherein said enzyme is alkaline phosphatase and said redox reaction system comprises X phosphate and nitroblue tetrazolium.

### Claim 18 (depends on 13)

Method of claim 13 comprising detecting said labelled antibody via a system using a leuko indicator.

### Claim 19 (depends on 13)

Method of claim 13 comprising detecting said label with an indigoid compound and a tetrazolium salt.

### Claim 20 (depends on 1)

Method of claim 1 wherein said first nucleic acid sequence is in a cell sample fixed to a solid support.

### Claim 21 (depends on 1)

Method of claim 1 wherein said first nucleic acid sequence is in a smear of tissue sample.

### Claim 22 (depends on 1)

Method of claim 1, wherein said hapten has been incorporated into said probe enzymatically.

### Claim 23 (depends on 22)

Method of claim 22, wherein said hapten is bound to a deoxy or ribonucleoside triphosphate moiety and said moiety has been incorporated into said probe enzymatically via an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, a reverse transcriptase and a terminal transferase.

### Claim 24 (depends on 1)

Method of claim 1, wherein said hapten has been incorporated into said probe photochemically.

### Claim 25 (depends on 24)

Method of claim 24, wherein said hapten is incorporated in the form of a photo-hapten.

### Claim 26 (depends on 1)

Method of claim 1, wherein said hapten has been incorporated into said probe chemically.

### Claim 27 (depends on 26)

Method of claim 26, wherein said hapten has been incorporated into said probe by incorporating a modified nucleoside phosphoamidite having a substitutable amino functional group into said probe as a protective group, removing said protective group, activating an ester, amide, or ether group of said hapten and reacting the activated hapten with said nucleoside phosphoamidite.

### Claim 28

Method for detection of a first nucleic acid sequence comprising:
(a) contacting said first nucleic acid with at least two oligonucleotide primers under conditions favoring hybridization between said first nucleic acid sequence and said primers, wherein the first primer comprises a first nucleotide sequence which is complementary to a part of a nucleic acid sequence of a DNA strand to be detected and wherein the second primer comprises a different second nucleotide sequence which is identical to a part of a nucleotide sequence of said DNA strand to be detected,
(b) treating said mixture from (a) with a polymerase and deoxyribonucleotides under conditions favoring nucleic acid polymerization and in the presence of at least one deoxyribonucleotide having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide via a bridge of at least 4 atoms length at a position thereon which does not participate in formation of hydrogen bonds with any other nucleotide,
(c) subjecting said mixture of (b) at least once to a cycle of denaturing, hybridizing and polymerizing according to steps (a) and (b),
(d) contacting said mixture from (c) with a labelled antibody which specifically binds to said steroid hapten, and
(e) detecting said labelled antibody as a detection of said first nucleic acid sequence.

### Claim 29 (depends on 28)

Method of claim 28 wherein said polymerase is a Thermus aquaticus DNA polymerase.

### Claim 30 (depends on 28)

Method of claim 28, wherein at least one of said primers is immobilized on a solid phase.

### Claim 31

Method for detection of a first nucleic acid sequence comprising:
(a) contacting said first nucleic acid with at least two oligonucleotide primers under conditions favoring hybridization between said first nucleic acid sequence and said primers, wherein the first primer comprises a first nucleotide sequence which is complementary to a part of a nucleic acid sequence to be detected and wherein the second primer comprises a different second nucleotide sequence which is identical to a part of said nucleotide sequence to be detected,
(b) treating said mixture from (a) with a polymerase and deoxyribonucleotides under conditions favoring nucleic acid polymerization,
(c) subjecting said mixture of (b) at least once to a cycle of denaturing, hybridizing and polymerizing according to steps (a) and (b),
(d) repeating the cycle according to step (c) in the presence of at least one deoxyribonucleotide having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide via a bridge of at least 4 atoms length at a position thereon which does not participate in formation of hydrogen bonds with any other nucleotide,
(e) contacting said mixture from (c) with a labelled antibody which specifically binds to said steroid hapten, and
(f) detecting said labelled antibody as a detection of said first nucleic acid sequence.

### Claim 32 (depends on 31)

Method of claim 31 wherein said polymerase is a Thermus aquaticus DNA polymerase.

### Claim 33 (depends on 31)

Method of claim 31, wherein at least one of said primers is immobilized on a solid phase.

### Claim 34

A deoxyribonucleotide or ribonucleotide having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide or ribonucleotide via a bridge of at least 4 atoms length at a position which does not participate in formation of hydrogen bonds with any other nucleotide.

### Claim 35

A deoxyribonucleotide or ribonucleotide triphosphate having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide or ribonucleotide via a bridge of at least 4 atoms length at a position which does not participate in formation of hydrogen bonds with any other nucleotide.