Sandwich hybridization method for nucleic acid detection

Claim 1

A method for detecting the presence of nucleic acid containing a gene or base sequence of interest comprising: a) providing the nucleic acid to be tested in a single strand form; b) contacting a biotinylated nucleic acid probe specific for a first gene or base sequence of said nucleic acid with an avidin coated solid phase support; c) reacting said nucleic acid with an excess of labeled nucleic acid probe specific for a second gene or base sequence of said nucleic acid and permitting any hybridization of said labeled probe and said nucleic acid to occur; d) reacting said nucleic acid with said biotinylated nucleic acid probe and permitting any hybridization of said biotinylated probe and said nucleic acid to occur; e) separating said unbound nucleic acid from said bound nucleic acid; and f) detecting the label associated with said bound nucleic acid or the decrease in the amount of label in solution for determining the presence of nucleic acid containing the gene or base sequence of interest.

Claim 2

A method for detecting the presence of nucleic acid containing a gene or base sequence of interest comprising: a) providing the nucleic acid to be tested in a single strand form; b) contacting a biotinylated nucleic acid probe specific for a first gene or base sequence of said nucleic acid with an avidin coated solid phase support; c) reacting said nucleic acid with an excess of labeled nucleic acid probe specific for a second gene or base sequence of said nucleic acid and permitting any hybridization of said labeled probe and said nucleic acid to occur; d) reacting said nucleic acid with said biotinylated nucleic acid probe and permitting any hybridization of said biotinylated probe and said nucleic acid to occur; e) separating said unbound nucleic acid from said bound nucleic acid; and f) detecting the label associated with said bound nucleic acid or the decrease in the amount of label in solution for determining the presence of nucleic acid containing the gene or base sequence of interest.

Claim 4

A method for detecting the presence of nucleic acid containing a gene or base sequence of interest comprising: a) providing the nucleic acid to be tested in ° single stranded form; b) further providing an avidin coated solid phase support which has been reacted with a first biotinylated nucleic acid probe to form a first solid phase species; c) contacting said first solid phase species with a nucleic acid probe linking means having a first portion which is reactive with at least some part of said first biotinylated nucleic acid probe and a second portion reactive with a first gene or base sequence of said nucleic acid and permitting any hybridization to occur to form a first mixture; d) reacting said nucleic acid with an excess of labeled nucleic acid probe specific for a second gene or base sequence of said nucleic acid and permitting any hybridization of said labeled probe and said nucleic acid to occur to form a second mixture; e) reacting said first mixture with said second mixture; f) separating solid phase immobilized nucleic acid from unbound nucleic acid; and g) detecting the label associated with said immobilized nucleic acid portion for determining the presence of nucleic acid containing the gene or base sequence of interest.

Claim 5

A method for detecting the presence of nucleic acid containing a gene or base sequence of interest comprising: a) providing the nucleic acid to be tested in ° single stranded form; b) further providing an avidin coated solid phase support which has been reacted with a first biotinylated nucleic acid probe to form a first solid phase species; c) contacting said first solid phase species with a nucleic acid probe linking means having a first portion which is reactive with at least some part of said first biotinylated nucleic acid probe and a second portion reactive with a first gene or base sequence of said nucleic acid and permitting any hybridization to occur to form a first mixture; d) reacting said nucleic acid with an excess of labeled nucleic acid probe specific for a second gene or base sequence of said nucleic acid and permitting any hybridization of said labeled probe and said nucleic acid to occur to form a second mixture; e) reacting said first mixture with said second mixture; f) separating solid phase immobilized nucleic acid from unbound nucleic acid; and g) detecting the label associated with said immobilized nucleic acid portion for determining the presence of nucleic acid containing the gene or base sequence of interest.

Claim 7

The method of any one of Claims 1 to 4 wherein the solid phase is selected from the group consisting of microparticles and macroscopic surfaces.

Claim 11

The method of any one of Claims 1 to 8 wherein the detecting step comprises detecting fluorescence.

Claim 12

The method of any one of Claims 1 to 8 wherein the detecting step comprises detecting chemiluminescence.

Claim 13

The method of any one of Claims 1 to 8 wherein the detecting step comprises detecting an isotopic label.

Claim 14

The method of any one of Claims 1 to 8 wherein the detecting step comprises detecting products of enzymatic labels.

Claim 15

The method of any one of Claims 1 to 8 wherein the detecting step comprises detecting light scatter.

Claim 16

The method of any one of Claims 1 to 4 wherein the detecting step comprises detecting by colorimetry.

Claim 17

The method of any one of Claims 1 to 14 wherein the providing step further comprises splitting double stranded nucleic acid into single stranded nucleic acid if said nucleic acid is deoxyribose nucleic acid.

Claim 18

A method for detecting the presence of nucleic acid containing a gene or base sequence of interest comprising: a) providing the nucleic acid to be tested in a single strand form; b) attaching a nucleic acid probe specific for a gene or base sequence of said nucleic acid to a nitrocellulose microparticle; c) reacting said nucleic acid with an excess of labeled nucleic acid probe specific for a first gene or base sequence of said nucleic acid and permitting any hybridization of said labeled probe and said nucleic acid to occur; d) reacting said nucleic acid with said nitrocellulose attached nucleic acid probe and permitting any hybridization to occur; e) separating said unbound nucleic acid from said bound nucleic acid. f) detecting the label associated with said bound nucleic acid or the decrease in the amount of label in solution for determining the presence of nucleic acid containing the gene or base sequence of interest.

Claim 19

A method for detecting the presence of nucleic acid containing a gene or base sequence of interest comprising: a) providing the nucleic acid to be tested in a single strand form; b) attaching a nucleic acid probe specific for a gene or base sequence of said nucleic acid to a nitrocellulose microparticle; c) reacting said nucleic acid with an excess of labeled nucleic acid probe specific for a first gene or base sequence of said nucleic acid and permitting any hybridization of said labeled probe and said nucleic acid to occur; d) reacting said nucleic acid with said nitrocellulose attached nucleic acid probe and permitting any hybridization to occur; e) separating said unbound nucleic acid from said bound nucleic acid. f) detecting the label associated with said bound nucleic acid or the decrease in the amount of label in solution for determining the presence of nucleic acid containing the gene or base sequence of interest.