Hybridization assay and means to be used in the assay

• US 5,256,535 A
• Filed: 06/16/1988
• Issued: 10/26/1993
• Est. Priority Date: 10/17/1986
• Status: Expired due to Term

• Current Assignee: Wallac Oy (Revvity, Inc.)
• Original Assignee: Wallac Oy (Revvity, Inc.)
• Inventors: Sund, Christian, Hurskainen, Pertti, Lovgren, Timo, Ylikoski, Jyrki, Dahlen, Patrik, Kwjatkowski, Marek
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): KIM, HYO
• Application Number:
  US07/207,159
• Time in Patent Office: 1,958 Days
• Field of Search: 436/546, 436/82, 435/6
• US Class Current: 435/6.13
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

A method for the detection of a nucleotide sequence of a nucleic acid in a sample. The method comprises the steps: (i) contacting under hybridization condition the single stranded form of the nucleotide sequence with a single stranded nucleic acid probe, in which plurality of rare earth metal chelate groups is covalently linked via a water-soluble polymer of non-nucleic acid structure to a nucleotide acid that as one of its strand has the nucleotide sequence to be detected and as the other strand the nucleotide sequence of the probe, and (ii) detecting the formation of double stranded nucleic acid. The plurality of rare earth metal chelate groups have at least one metal ion selected from the group consisting of Eu³⁺, Sm³⁺, Tb³⁺ and Dy³⁺ as the chelated rare earth metal. The probes as given above are also claimed.

Claims

1. A hybridization assay method for the detection of a nucleotide sequence of a nucleic acid in a sample, said method comprising the steps:

(i) contacting under hybridization conditions the single stranded form of the sample nucleotide sequence with a single stranded nucleic acid probe, wherein;

a) said probe has a nucleotide sequence complementary to the sequence to be detected,

b) said probe has a water-soluble polymer bearing a plurality of chelating structures covalently linked to said polymer, wherein said water-soluble polymer is of non-nucleic acid structure and is covalently linked to said nucleotide sequence complementary to the sequence to be detected, and

c) said probe has a plurality of rare earth metal ions that together with said chelating structures forms a plurality of chelate groups covalently bound to said water-soluble polymer, and said rare earth metal is selected from the group consisting of Eu³⁺, Tb³⁺, Sm³⁺, and Dy³⁺, wherein said probe and covalently bound chelate groups are stable under hybridization assay conditions;

(ii) allowing a nucleotide sequence to be detected in said sample and the nucleotide sequence of said probe to form a double stranded nucleic acid; and

(iii) detecting the formation of double stranded nucleic acid containing said probe by measuring the time-resolved fluorescence from the rare earth metal ions incorporated as a chelate in said double stranded nucleic acid.

2. The method of claim 1, wherein the said at least one metal ion is selected from the group consisting of Eu³⁺ and Tb³⁺.

3. The method of claim 1, wherein the water-soluble polymer is selected from the group of polymers having a plurality of OH-- or amino groups.

4. The method of claim 3, wherein said water-soluble polymer having a plurality of OH groups is selected from the group consisting of polymers having alcohol, phenol and carboxy groups.

5. The method of claim 3, wherein the polymer is selected from the group of polymers consisting of polymers having a plurality of amino groups.

6. The method of claim 3, wherein the water-soluble polymer is selected from the group of polymers consisting of polyvinylamines, polyethyleneimines, polylysine, polysacharides, polyacrylamides, and derivatized forms of these polymers exhibiting the said plurality of OH--or amino groups.

7. The method of claim 1, wherein at least two nucleotide sequences complementary to the sequence to be detected are bound to the water-soluble polymer molecule.

8. The method of claim 1, wherein at least two water-soluble polymer molecules are bound to the nucleotide sequence complementary to the nucleotide sequence to be detected.

9. Nucleotide sequence to which a plurality of lanthanide chelate groups are bound covalently via a water-soluble polymer, said lanthanide being non-radioactive and selected from a group consisting of Dy³⁺, Sm³⁺, Eu³⁺ and Tb³⁺, preferably from group Eu³⁺ and Tb³⁺.