Hybridisation probes

Claim 2

A method as claimed in claim 1 wherein the detectable first nucleotide probe and second nucleotide probe are hybridised to each side of a potential variant sequence in a target sequence the said probes being subjected to a linkage reaction which comprises introducing a nucleotide(s) complementary to either the normal or the suspected variant sequence and subjecting the nucleotide(s) to ligation; followed by detection of any hybrid obtained

Claim 3

A method as claimed in claim 2 wherein detection is effected by subjecting any hybrid obtained to selective denaturing whereby any hybrid present, in which linkage of the detectable first nucleotide probe to the second nucleotide probe has not been effected, is denatured.

Claim 4

A method as claimed in claim 2 or claim 3 wherein the introduction of the said nucleotide(s) is effected by the use of DNA polymerase.

Claim 6

A method as claimed in claim 1 in which a split probe would be formed following hybridisation, with a complementary target base sequence, any hybrid obtained being subjected to a linking reaction for linking the detectable first nucleotide probe to the second nucleotide probe, and the hybrid thus obtained being subjected to appropriate treatment whereby any hybrid present, in which linkage of the detectable first nucleotide probe to second nucleotide probe has not been effected, is denatured whereas no denaturation is effected for a perfectly complementary detectable linked oligonucleotide-target sequence hybrid.

Claim 7

A method as claimed in claim 1 wherein, adjacent segments of the target base sequence are subjected to hybridisation with a detectable first nucleotide probe and with a second nucleotide probe such that a split probe hybrid would be formed with a complementary target sequence, the hybrid obtained being subjected to selective denaturing whereby to denature whichever of the first and second nucleotide probes is hybridised to a portion of the target base sequence with one or more non-complementary nucleotides the hybrid obtained subsequently being subjected to a linking reaction whereby to link together the detectable first nucleotide probe to the second nucleotide probe, and subjecting the hybrid obtained to selective denaturation, whereby to discriminate between a target sequence complementary to the first and second nucleotide probes and a target sequence with one or more nucleotides non-complementary to one of the first and second nucleotide probes.

Claim 8

A method as claimed in claim 1 for the analysis of translocations in a target base sequence wherein the detectable first nucleotide probe is adapted to hybridise across a region of potential translocation(s) and the non-detectable second nucleotide probe is adapted to hybridise to sequences adjacent to said potential translocation(s), the non-detectable second nucleotide probe carrying one member of a binding pair, the method comprising formation of a split probe hybrid which is subjected to linking to form a linked probe hybrid which hybrid is then denatured.

Claim 9

A method as claimed in claim 1 wherein each of the detectable first nucleotide probe and second nucleotide probe carry a moiety such that after hybrid formation, and denaturation where appropriate, a signal is only detectable if a nucleotide sequence is obtained which carries both the moiety attached to the detectable first nucleotide probe and the moiety attached to the second nucleotide probe.

Claim 10

A method as claimed in claim 1 for discriminating between alternative nucleotide sequences in which a target sequence is subjected to hybridisation with more than two oligonucleotide probes such that where a complementary target sequence is present a split probe hybrid is obtained, the detectable first nucleotide probe being a terminal oligonucleotide probe which is detectable and the second nucleotide probe being the other terminal oligonucleotide probe which has attached thereto one member of a binding pair, individual other oligonucleotide probes(s) being hybridised separately but adjacently to a contiguous target sequence between the said terminal oligonucleotide probes, the hybrid obtained is then subjected to selective denaturation whereby to denature any oligonucleotide probe hybridised to the target sequence across a base pair mismatch, and the individual oligonucleotide probes linked to join the detectable oligonucleotide probe to the oligonucleotide probe having one member of a binding pair attached thereto to form a linked probe hybrid which is then denatured and contacted with the other member of the binding pair whereby the detectable linked probe nucleotide sequence including the said binding pair may be separated from other nucleotide sequences.

Claim 11

A split probe hybrid comprising a detectable first nucleotide probe and a second nucleotide probe hybridised to adjacent segments of a target base sequence, the detectable first nucleotide probe being capable of linkage to the second nucleotide probe, characterised in that the detectable first nucleotide probe and/or the second nucleotide probe are hybridized to either side of a variant sequence associated with a disease state or to the corresponding normal sequence; or are hybridised to the target base sequence such that a variant base sequence associated with a disease state therein is at the terminal end of one of said probes, which terminal end is contiguous with the other of said probes or are hybridised to the corresponding normal sequence.

Claim 12

A kit for discriminating between alternative nucleotide sequences according to the method of claim 1, which comprises a detectable first nucleotide probe and a second nucleotide probe, each probe having a nucleotide sequence homologous to adjacent segments of a target sequence, a potential variant sequence being present in one of said segments or therebetween; the detectable first nucleotide probe and/or the second nucleotide probe being such that a potential variant sequence is at the terminal end of one of said probes, which terminal end is contiguous with the other of said probes or the potential variant sequence is present between said probes, the kit additionally containing a reagent(s) for linking said probes.

Claim 13

A kit as claimed in claim 12 which contains a detectable first nucleotide probe and second nucleotide probe for detecting a point mutation associated with a disease state as well as a detectable first nucleotide probe and a second nucleotide probe for detecting the corresponding normal sequence in which the point mutation was absent.