Diagnosis of gene abnormalities by restriction mapping using a sandwich hybridization format

Claim 1

1 . A method for determining the presence of a specific base pair sequence occuring within a nucleic acid strand in an aqueous sample comprising: a) providing an insolubilized nucleic acid probe homologous to a first base pair sequence on one side of said specific base pair sequence; b) further providing a labeled nucleic acid probe homologous to a second base pair sequence on the other side of said specific base pair sequence; c) contacting said nucleic acid strand either with a restriction endonuclease which will cleave said nucleic acid strand at said specific base pair sequence if present but not at any other site between or along said first and second base pair sequences whereby said nucleic acid strand is digested into at least two portions if said specific base pair sequence is present or with a restriction endonuclease which will not cleave said nucleic acid strand at said specific base pair sequence if present nor at any other site between or along said first and second base pair sequences whereby said first and second base pair sequences remain connected if said specific base pair sequence is present; d) denaturing said nucleic acid strand into single stranded nucleic acid after said contacting step; e) reacting said single stranded nucleic acid with said insolubilized nucleic acid probe and said labeled nucleic acid probe under conditions which permit hybridization to occur; f) separating said insolubilized probe from said aqueous sample; and g) determining the presence of said specific base pair sequence on the basis of whether or not said labeled probe is associated with said immobilized probe or with said aqueous sample following said separation step.

Claim 5

The method as provided in any one of claims 1 to 3 wherein said specific base pair sequence is associated with a genetic abnormality in turn associated with a disease condition whereby the determination of the presence of said genetic abnormality constitutes diagnostic evidence of said disease.

Claim 6

The method as provided in any one of claims 1 to 4 wherein said insolbilized nucleic acid probe is immobilized by attachment to a solid phase selected from the group consistingof microbeads, latex particles, plastic surfaces, glass surfaces and porous surfaces; and said label is selected from the group consisting or radioisotopes, fluorophores, chromophores, enzymes, magnetic particles, light scattering particles and reflective metallic particles.

Claim 7

The method as provided in any one of claims 1 to 5 wherein said insolubilized nucleic acid probe is immobilized on nitrocellulose paper and said label is 131 I , 32 P 125 I , of 35 S .

Claim 10

The method as provided in any one of claims 1 to 8 wherein said separation step is selected from the group consisting of washing and centrifuging.

Claim 11

The method as provided in any one of claims 1 to 9 wherein at least one of said first or second nucleic acid probes is an oligodeoxynucleotide homologous to a base pair sequence on said sample nucleic acid strand which includes said restriction site and wherein the binding properties of said oligodeoxynucleotide with its homologous base pair sequence will be detectably altered with restriction endonuclease digestion at said specific base pair sequence.

Claim 12

A method for determining the presence of a specific base pair sequence occurring within a nucleic acid strand in an aqueous sample comprising: a) providing a first insolubilized nucleic acid probe homologous to a first base sequence on one side of said specific base pair sequence; b) further providing a second insolubilized nucleic acid probe homologous to a second base sequence on the other side of said specific base pair sequence; c) contacting said nucleic acid with a restriction endonuclease which will cleave said nucleic acid at said specific base pair sequence if present but not at any other site between or along said first and second base pair sequences whereby said nucleic acid is digested into at least two portions if said specific base pair sequence is present; d) denaturing said nucleic acid into single stranded nucleic acid after said contacting step; e) reacting said single stranded nucleic acid with said first and second insolubilized nucleic acid probes under conditions which permit hybridization to occur; f) determining the presence of said specific base pair sequence on the basis of agglutination or changes in agglutination settling rates.

Claim 13

A method for determining the presence of a specific base pair sequence occurring within a nucleic acid strand in an aqueous sample comprising: a) providing a first insolubilized nucleic acid probe homologous to a first base sequence on one side of said specific base pair sequence; b) further providing a second insolubilized nucleic acid probe homologous to a second base sequence on the other side of said specific base pair sequence; c) contacting said nucleic acid with a restriction endonuclease which will cleave said nucleic acid at said specific base pair sequence if present but not at any other site between or along said first and second base pair sequences whereby said nucleic acid is digested into at least two portions if said specific base pair sequence is present; d) denaturing said nucleic acid into single stranded nucleic acid after said contacting step; e) reacting said single stranded nucleic acid with said first and second insolubilized nucleic acid probes under conditions which permit hybridization to occur; f) determining the presence of said specific base pair sequence on the basis of agglutination or changes in agglutination settling rates.