Hybridization method and probes therefor

Claim 1

A method for discriminating between two complementary nucleotide sequences differing by as little as 13 nucleotides which comporises:- (a) under conditions which promote or favour hybridisation between two complementary oligonucleotide strands, contacting the nucleotide sequence (after appropriate treatment to render it single stranded) to be identified or determined with an oligonucleotide-signalling complex probe comprising a single stranded oligonucleotide of 8 to 30 nucleotide units in length and having covalently attached thereto, at a sterically tolerant site, a residue having a molecular weight of at least 1,000 daltons, which residue is capable of producing a non-radioisotopic signal, the oligonucleotide in the oligonucleotide-signal complex having a defined sequence which is complementary to a sequence to be identified or determined but which is not complementary to an alternative sequence; (b) separating the probe:test nucleic acid hybrid from the unhybridized probe; and (c) producing or observing the signal resulting from the hybridised oligonucleotide-signalling complex.

Claim 2

A method for discriminating between two complementary nucleotide sequences differing by as little as 13 nucleotides which comporises:- (a) under conditions which promote or favour hybridisation between two complementary oligonucleotide strands, contacting the nucleotide sequence (after appropriate treatment to render it single stranded) to be identified or determined with an oligonucleotide-signalling complex probe comprising a single stranded oligonucleotide of 8 to 30 nucleotide units in length and having covalently attached thereto, at a sterically tolerant site, a residue having a molecular weight of at least 1,000 daltons, which residue is capable of producing a non-radioisotopic signal, the oligonucleotide in the oligonucleotide-signal complex having a defined sequence which is complementary to a sequence to be identified or determined but which is not complementary to an alternative sequence; (b) separating the probe:test nucleic acid hybrid from the unhybridized probe; and (c) producing or observing the signal resulting from the hybridised oligonucleotide-signalling complex.

Claim 5

A method as claimed in any one of the preceding claims effective to discriminate between two complementary nucleotide sequences differing by as little as a single nucleotide.

Claim 6

A method as claimed in any one of the preceding claims wherein the residue which is capable of producing a non-radioisotopic signal comprises a proteinaceous moiety.

Claim 7

A method as claimed in any one of the preceding claims wherein the oligonucleotide signalling complex probe comprises a single stranded oligonucleotide sequence having a detection system covalently attached thereto via a spacer group, said detection system comprising a system for producing an enzymatically activated production of colour change, the system being linked to the spacer group by a direct covalent link or by a specific binding pair.