Process for the detection of nucleic acids
AssigneeBoehringer Mannheim GmbH
InventorsHans-Joachim Holtke, Rudolf Seibl, Gudrun Schmitz, Hans Robert Scholer, Christoph Kessler, Ralf Mattes
Life of this patent
Estimating the term…
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 vie 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.
Claims35
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, said probe characterized as a second nucleic acid sequence having bound thereto at least one steroid hapten, said steroid hapten being bound to said second nucleic acid sequence to a position thereon which does not participate in formation of hydrogen bonds with said first sequence via a bridge of at least 4 atoms length, 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.
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 said 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 deoxyribonucleotides having bound thereto at least one steroid hapten, said steroid hapten being bound to said deoxyribonucleotides via a bridge of at least 4 atoms length to 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 labeled antibody which specifically binds to said steroid hapten, and
- (e) detecting said labeled antibody thereby detecting said first nucleic acid sequence.
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 said 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 pan 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 deoxyribonucleotides having bound thereto at least one steroid hapten, said steroid hapten being bound to said deoxyribonucleotides via a bridge of at least 4 atoms length to a position thereon which does not participate in formation of hydrogen bonds with any other nucleotide,
- (e) contacting said mixture from (c) with a labeled antibody which specifically binds to said steroid hapten, and
- (f) detecting said labeled antibody thereby detecting said first nucleic acid sequence.
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 said 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, said steroid hapten being bound to said deoxyribonucleotide via a bridge of at least 4 atoms length to a position thereon which does not participate in formation of hydrogen bonds with nay 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.
A deoxyribonucleotide or ribonucleotide having bound thereto at least one steroid hapten, said steroid hapten being bound to said deoxyribonucleotide or ribonucleotide via a bridge of at least 4 atoms length to a position which does not participate in formation of hydrogen bonds with any other nucleotide.
A deoxyribonucleotide or ribonucleotide triphosphate having bound thereto at least one steroid hapten, said steroid hapten being bound to said deoxyribonucleotide or ribonucleotide via a bridge of at least 4 atoms length to a position which does not participate in formation of hydrogen bonds with any other nucleotide.
Dependencies are inferred from the claim text.