Quantitative detection of macromolecules with fluorescent oligonucleotides

• US 5,445,935 A
• Filed: 11/23/1992
• Issued: 08/29/1995
• Est. Priority Date: 11/23/1992
• Status: Expired due to Fees

• Current Assignee: R-P Technologies Incorporated
• Original Assignee: R-P Technologies Incorporated
• Inventors: Royer, Catherine A.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): SCHWARTZMAN, ROBERT A
• Application Number:
  US07/980,283
• Time in Patent Office: 1,009 Days
• Field of Search: 435/6, 436/94, 436/501, 436/503, 436/6
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 2021/6463 Optics
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters

Abstract

A method is described by which the association between an oligonucleotide labeled by attachment of a fluorophore and another macromolecule such as a protein or nucleic acid may be determined quantitatively in solution accurately and with high sensitivity. In the performance of this method the polarization of fluorescence of an extrinsic fluorescence probe that is covalently coupled to the oligonucleotide is determined. Changes in fluorescence polarization are related directly to the degree of association between the labeled oligonucleotide and another macromolecule and may be used to quantify the association. Because of its high sensitivity and accuracy, this method may be used to make reliable quantitative measurements of very small amounts of complexes formed between labeled oligonucleotides and proteins, nucleic acids or other macromolecules. The method also allows the accurate calculation of important biochemical parameters such as dissociation constants. The method, which is rapid and solution-based, has a broad range of applications in biochemistry, genetic cloning and molecular biology, as well as in clinical diagnostics.

Claims

1. A method for measuring the binding of a polynucleotide to a macromolecule to form a complex which comprises:

i) labelling said polynucleotide with a fluorescent label;

ii) obtaining a first measurement of the polarization of the fluorescent emission from said label;

iii) contacting said labelled polynucleotide with said macromolecule, under conditions such that the concentration of said labelled polynucleotide ranges from 10 picomolar to 1 nanomolar;

iv) obtaining a second measurement of the polarization of the fluorescent emission from said label;

v) comparing said first measurement with said second measurement; and

vi) detecting formation of said complex by observing an increase in the polarization measured in step (iv) compared to the polarization measured in step (ii);

wherein said measurements of fluorescence polarization are made using an apparatus having a component selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer, an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.

2. A method as recited in claim 1, wherein said label is fluorescein.

3. A method as recited in claim 1, wherein said polynucleotide is a single-stranded oligodeoxyribonucleotide of length less than or equal to 40 bases.

4. A method as recited in claim 3, wherein said macromolecule is a second polynucleotide.

5. A method as recited in claim 1, wherein said polynucleotide is a double-stranded oligodeoxyribonucleotide of length less than or equal to 40 basepairs.

6. A method as recited in claim 1, wherein said macromolecule is a protein.

7. A method as recited in claim 1, wherein said macromolecule is present at a concentration ranging from 10 picomolar to 1 nanomolar.

8. The method of claim 7, which further comprises the following steps:

vii) contacting unlabeled polynucleotide at a concentration of 10 picomolar to 1 nanomolar with the labelled polynucleotide and macromolecule of step (iii), said unlabeled polynucleotide having the same nucleotide sequence as said labelled polynucleotide;

viii) obtaining a third measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide

ix) comparing said third measurement to said first and second measurements,

wherein the binding is measured by observing a change in the polarization measured in each of steps (ii), (iv) and (viii).

9. A method for detecting the presence of wild-type or mutant gene in a patient which comprises:

i) obtaining a sample of DNA from the patient;

ii) obtaining a first measurement of the polarization of a fluorescent emission from a fluorescently-labelled oligonucleotide probe which is capable of binding exclusively to either wild-type sample DNA or mutant DNA;

iii) contacting said probe at a concentration of 10 picomolar to 1 nanomolar under conditions such that the probe binds exclusively to either wild-type DNA or mutant DNA;

iv) obtaining a second measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide; and

v) comparing said second measurement to said first measurement;

wherein a change in polarization measured in step (ii) and step (iv) indicates the presence of the wild-type or mutant gene;

wherein said measurements of fluorescence polarization are made using an apparatus selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer, an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.

10. A method for determining the amount of a DNA binding protein which comprises:

i) fluorescently labelling an oligonucleotide having a nucleotide sequence recognized by a DNA-binding protein, wherein said oligonucleotide may be either double-stranded or single stranded;

ii) obtaining a sample containing the DNA-binding protein to be tested;

iii) obtaining a first measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide;

iv) contacting said fluorescently-labelled oligonucleotide with said sample under conditions such that the concentration of said fluorescently-labelled polynucleotide ranges from 10 picamolar to 1 nanomolar;

v) obtaining a second measurement of the polarization of the fluorescent emission from said fluorescently-labelled oligonucleotide;

vi) comparing said second measurement to said first measurement;

wherein the amount of change between the first and second measurements determines the amount of DNA binding protein, wherein said measurements of fluorescence polarization are made using an apparatus selected from the group consisting of an apparatus having a Xenon arc lamp light source and a UV grade film polarizer an apparatus having a Xenon arc lamp light source coupled by a fiber optic cable to a sample compartment and a UV grade film polarizer mounted directly on the sample compartment, and an apparatus having a laser light source.