Method for the sensitive detection of nucleic acids

Claim 1

A method for the specific detection of a nucleic acid in a sample comprising the steps of: a) first reacting the sample with at least one labelled mononucleoside triphosphate and at least one enzyme which catalyzes the production of a labelled nucleic acid B, which contains said labelled mononucleoside triphosphate, to form a reaction mixture; wherein said sample is reacted in the presence of a primer P1, which is sufficiently complementary to a part of a template nucleic acid to hybridize specifically to said part of the template nucleic acid and is elongated by the enzyme to form said nucleic acid B which incorporates the labelled mononucleoside triphosphate and which is sufficiently complementary to at least part of the template nucleic acid to hybridize specifically to the template nucleic acid, b) then non-thermally denaturing any nucleic acids in the reaction mixture, c) then hybridizing the nucleic acid B with a nucleic acid probe C which contains at least one immobilizable group and which is sufficiently complementary to the nucleic acid B to hybridize specifically with nucleic acid B, d) immobilizing the nucleic acid probe C on a solid phase, e) then separating the solid phase from the reaction mixture, and f) thereafter detecting a nucleic acid hybrid D formed from the labelled nucleic acid B and nucleic acid probe C by detecting any labeled nucleic acid bound to the solid phase, wherein steps c) and d) are performed simultaneously and wherein said immobilizable group is different from the label on said labelled mononucleoside triphosphate.

Claim 2

A method for the specific detection of a nucleic acid in a sample comprising the steps of: a) first reacting the sample with at least one labelled mononucleoside triphosphate and at least one enzyme which catalyzes the production of a labelled nucleic acid B, which contains said labelled mononucleoside triphosphate, to form a reaction mixture; wherein said sample is reacted in the presence of a primer P1, which is sufficiently complementary to a part of a template nucleic acid to hybridize specifically to said part of the template nucleic acid and is elongated by the enzyme to form said nucleic acid B which incorporates the labelled mononucleoside triphosphate and which is sufficiently complementary to at least part of the template nucleic acid to hybridize specifically to the template nucleic acid, b) then non-thermally denaturing any nucleic acids in the reaction mixture, c) then hybridizing the nucleic acid B with a nucleic acid probe C which contains at least one immobilizable group and which is sufficiently complementary to the nucleic acid B to hybridize specifically with nucleic acid B, d) immobilizing the nucleic acid probe C on a solid phase, e) then separating the solid phase from the reaction mixture, and f) thereafter detecting a nucleic acid hybrid D formed from the labelled nucleic acid B and nucleic acid probe C by detecting any labeled nucleic acid bound to the solid phase, wherein steps c) and d) are performed simultaneously and wherein said immobilizable group is different from the label on said labelled mononucleoside triphosphate.

Claim 24

A method for specifically detecting a nucleic acid in a sample comprising the steps of: a) adding mononucleoside triphosphates and at least one enzyme which catalyzes the production of a nucleic acid B by incorporating said mononucleoside triphosphates, to the sample to form a reaction mixture, wherein at least one of the mononucleoside triphosphates is detectably labelled, b) thereafter denaturing any nucleic acids in the reaction mixture, c) then hybridizing the nucleic acid B with a nucleic acid probe C which contains at least one immobilizable group, d) immobilizing the nucleic acid probe C on a solid phase, e) separating the reaction mixture from the solid phase, and f) detecting any labeled nucleic acid bound to the solid phase, wherein steps c) and d) are performed simultaneously.

Claim 25

A method for specifically detecting a nucleic acid in a sample comprising the steps of: a) adding mononucleoside triphosphates and at least one enzyme which catalyzes the production of a nucleic acid B by incorporating said mononucleoside triphosphates, to the sample to form a reaction mixture, wherein at least one of the mononucleoside triphosphates is detectably labelled, b) thereafter denaturing any nucleic acids in the reaction mixture, c) then hybridizing the nucleic acid B with a nucleic acid probe C which contains at least one immobilizable group, d) immobilizing the nucleic acid probe C on a solid phase, e) separating the reaction mixture from the solid phase, and f) detecting any labeled nucleic acid bound to the solid phase, wherein steps c) and d) are performed simultaneously.