Improved sandwich hybridisation technique for the detection of nucleotide sequences

Claim 3

A method according to claim 1, wherein the immobilised polynucleotide is covalently bonded to the resin beads by a diazotisation reaction between reactive groups on the resin and free amino groups on the polynucleotide.

Claim 4

A method according to claim 1, wherein the immobilised polynucleotide is covalently bonded to the resin particles or beads by carbodiimide coupling between the phosphate groups at the 5' end of the polynucleotide and reactive groups on the resin.

Claim 5

A method according to claim 1, 2 or 3, wherein the polynucleotide probe comprises a radioactive marker or label.

Claim 6

A method according to claim 1, 2 or 3, wherein the polynucleotide probe comprises an enzyme marker or label.

Claim 8

A method according to claim 6, as applied to the detection of the. abnormal human (sickle cell) βlobin gene, which comprises digesting the gene with a Dde I restriction endonuclease to produce a restriction digest containing as a component a 381 bp restriction fragment containing the abnormal sixth codon which is a characteristic of the abnormal gene, contacting the digest containing that fragment under hybridisation conditions with (a) said immobilised single- stranded polynucleotide covalently bonded to solid resin beads, said single-stranded polynucleotide being hybridisable with a first section of said fragment, and (b) with a labelled single-stranded polynucleotide probe hybridisable with a second section of said fragment, wherein one of said single-stranded polynucleotide probe and said labelled single- stranded polynucleotide is or comprises a 180 bp polynucleotide sequence obtainable by Dde I digestion of normal human p-globin gene and complementary to a first section of said 381 bp fragment, and the other is or comprises a 201 bp fragment obtainable by Dde I digestion of normal β-globin gene and complementary to a second, different, section of said 381 bp fragment, separating the beads carrying the resulting immobilised hybridisation duplex comprising the immobilised single-stranded polynucleotide, the said restriction fragment, and said labelled polynucleotide probe, and determining the presence of the marker on the separated duplex.

Claim 9

A method according to claim 7, wherein the said 180 bp polynucleotide sequence is replaced by a 341 bp fragment containing a sequence of 175 bp identical to that of the first 175 bp of said 180 bp fragment, said 341 bp fragment being obtainable by Hinf I digestion of normal human β-globin gene.

Claim 10

A method according to claim 8, wherein the said 341 bp fragment is obtained by the Hinf I digestion of plasmid β-F5, plasmid β-F5 itself being obtained by inserting the human P-globin gene into the Bam H1 site of vector pAT153.

Claim 12

A diagnostic kit for the detection of a given nucleotide sequence in a target polynucleotide, comprising as a first component an immobilised polynucleotide reagent comprising solid beads of a synthetic resin having a polynucleotide comprising a single-stranded polynucleotide sequence complementary to a first section of the nucleotide sequence to be detected covalently bonded to said particles or beads, and as a second component, a polynucleotide probe comprising a single-stranded nucleotide sequence complementary to a second, different section of the nucleotide sequence to be detected, and labelled with a marker, characterised in that said beads consist of N,N'-methylene bisacrylamide cross-linked allyl dextran (Sephacryl®), said beads having a size in the range 5 to 50 µm.

Claim 13

A diagnostic kit for the detection of a given nucleotide sequence in a target polynucleotide, comprising as a first component an immobilised polynucleotide reagent comprising solid beads of a synthetic resin having a polynucleotide comprising a single-stranded polynucleotide sequence complementary to a first section of the nucleotide sequence to be detected covalently bonded to said particles or beads, and as a second component, a polynucleotide probe comprising a single-stranded nucleotide sequence complementary to a second, different section of the nucleotide sequence to be detected, and labelled with a marker, characterised in that said beads consist of N,N'-methylene bisacrylamide cross-linked allyl dextran (Sephacryl®), said beads having a size in the range 5 to 50 µm.

Claim 14

A diagnostic kit according to claim 11 for the detection of the abnormal human (sickle cell) β-globin gene, characterised in that one of said single stranded polynucleotide sequences is or comprises a 180 bp polynucleotide sequence obtainable by Dde I digestion of normal human P-globin gene, and the other is or comprises a 201 bp fragment obtainable by Dde I digestion of normal β-globin gene.

Claim 15

A kit according to claim 12, modified in that the said 180 bp polynucleotide sequence is replaced by a 341 bp fragment obtainable by Hinf I digestion of normal human β-globin gene, said 341 bp fragment containing a sequence of 175 bp identical to the first 175 bp of said 180 bp fragment.

Claim 16

A kit according to claim 13, wherein said 341 bp fragment is a product obtained by Hinf I digestion of plasmid P-F5.

Claim 17

A k i t according to claim 14, wherein said 201 bp fragment is a product obtained by Mst II digestion of plasmid p-F5.

Claim 18

A kit according to any one of claims 10-15 wherein the said marker is an enzyme.

Claim 19

A kit according to claim 16, the kit also contains, as a third component, means for detecting the enzyme marker, said means comprising a substrate for said enzyme.