Quantitative detection of macromolecules with fluorescent oligonucleotides

• US 5,756,292 A
• Filed: 08/17/1995
• Issued: 05/26/1998
• 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): Houtteman, Scott W.
• Application Number:
  US08/516,331
• Time in Patent Office: 1,013 Days
• Field of Search: 435/6, 935/86, 250/372, 356/32, 356/33, 356/34, 356/35.5, 356/51, 356/327, 356/364, 356/366, 356/367, 356/368
• 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 is in the range of 10 picomolar to 10 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 polarization measurements are made using an apparatus comprising a high intensity light source as an illumination means wherein a sample chamber containing said polynucleotide and macromole is coupled to the high intensity light source by a fiber optic cable.

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 1, wherein said polynucleotide is a double-stranded oligodeoxyribonucleotide of length less than or equal to 40 basepairs.

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

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

7. A method as recited in claim 1, wherein said polynucleotide is present at a concentration of less than or equal to 1 nanomolar.

8. A method as recited in claim 1, wherein said macromolecule is present at a concentration of less than or equal to 1 nanomolar.

11. The method of claim 8, which further comprises:

vii) adding a second aliquot of unlabelled 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 under said conditions favorable to binding; and

vi) comparing said measurement to said first and second measurements.

23. The method of claim 1, wherein the concentration of said labelled oligonucleotide is in the range of 20 picomolar to 5 nanomolar.

25. The method of claim 1, wherein said fluorescent label is coumarin, eosin, fluorescein or a dansyl derivative.

9. A method for detecting the presence of a wild-type or of a mutant DNA sequence which comprises:

i) obtaining a sample of DNA from a patient;

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

iii) contacting said fluorescently-labelled oligonucleotide with said DNA under conditions such that the concentration of said fluorescently-labelled oligonucleotide is in the range of 10 picomolar to 10 nanomolar and such that the oligonucleotide selectively binds to either the wild-type or the mutant DNA present in the sample;

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 wild-type or mutant DNA;

wherein said polarization measurements are made using an apparatus comprising a high intensity light source as an illumination means wherein a sample chamber containing said polynucleotide and macromolecule is coupled to the high intensity light source by a fiber optic cable.

26. The method of claim 9, wherein said fluorescent label is coumarin, eosin, fluorescein or a dansyl derivative.

30. A method as recited in claim 9, wherein said fluorescently-labeled oligonucleotide has the nucleotide sequence of the wild-type gene and said conditions are favorable to binding to wild-type sequences in said DNA of said patient.

10. A method for diagnosing variation in the amount or nature of a DNA binding protein which comprises:

i) obtaining a first measurement of the polarization of the fluorescent emission from a fluorescently-labelled oligonucleotide, wherein said oligonucleotide has a nucleotide sequence recognized by a DNA-binding protein, wherein said oligonucleotide may be either double-stranded or single stranded;

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

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

iv) comparing said second measurement to said first measurement; and

v) diagnosing a variation in the amount or nature of said DNA binding protein by observing a change in the polarization measured in step (i) compared to the polarization measured in step (iii);

wherein said polarization measurements are made using an apparatus comprising a high intensity light source as an illumination means wherein a sample chamber containing said oligonucleotide and said DNA binding protein is coupled to the high intensity light source by a fiber optic cable.

27. The method of claim 10, wherein said fluorescent label is coumarin, eosin, fluorescein or a dansyl derivative.

12. An apparatus for measuring the polarization of fluorescent emission which comprises:

a box for the exclusion of light, in which is placed;

an excitation means for producing high intensity polarized excitation light having a narrow range of wavelengths;

a sample chamber illuminated by said polarized light wherein said sample chamber is coupled to the high intensity excitation means by a fiber optic cable;

polarizing means for imposing polarization upon fluorescence emission from said sample chamber to obtain both an emission polarized parallel to the plane of polarization of the excitation light and an emission polarized perpendicular to the plane of polarization of the excitation light, wherein said means comprise either a plurality of emission polarizers, or a single, rotatable polarizer, and wherein said polarizing means are of a film type;

detection means for detecting said fluorescence emission; and

emission lenses for focussing said fluorescent emission upon said detection means.

14. The apparatus of claim 12, wherein said illumination means is a xenon arc lamp.

13. An apparatus for measuring the polarization of fluorescent emission which comprises:

a box for the exclusion of light in which is placed a high intensity illumination means so as to illuminate a sample chamber, through a fiber optic cable, a collimating lens and a focusing lens, and further through a first polarizing means for plane polarizing the light from said illumination means, said lenses and first polarizing means, being mounted in a line connecting said illumination means and said sample chamber, and further said sample chamber being mounted between a second and a third polarizing means, said second and third polarizing means being placed so that the line between them and including said sample chamber is perpendicular to the light path from said illumination means to said sample chamber, said polarizing means being mounted so that the plane of polarization of said second polarizing means is parallel to the plane of polarization of said first polarizing means and also so that the plane of polarization of said second polarizing means is perpendicular to the plane of polarization of said third polarizing means and in which box is also placed detection means for the collection and quantitation of the amount of light which passes through said polarizing means from said sample chamber to said detecting means;

wherein each of said polarizing means is a film polarizer.

15. The apparatus of claim 13, wherein said collimating lens and said focusing lens are substituted by a fiber optic cable.

16. The apparatus of claim 13, wherein said illumination means is a laser and each of said collimating lens, said focusing lens and said first polarizing means are not present.

17. The apparatus of claim 16, wherein said laser is a tunable dye laser or a solid state laser.

18. The apparatus of claim 16, wherein each of said polarizing means are ultraviolet grade film polarizers.

19. The apparatus of claim 13, wherein the length of the light path from said illumination means to said detection means is 20 centimeters or less.

20. An apparatus for measuring the polarization of fluorescent emission which comprises:

A box for the exclusion of light in which is placed a high intensity illumination means so as to illuminate a sample chamber through a fiber optic cable, an excitation lens and further through an excitation filter, wherein said excitation filter passes only a narrow range of wavelengths of light, and further through a fixed means of plane polarizing light which shines on said sample chamber, such that each of these elements are mounted in the light path from said illuminating means to said sample chamber, said sample chamber being mounted relative to a rotatable polarizing means, which plane polarizes light which passes through said rotatable plane polarizing means, so that a path from said illumination means through said sample chamber to said rotatable polarizing means forms a right angle with the sample chamber at the vertex and in which box is also placed an emission filter interposed between the sample chamber and said rotatable plane polarizing means, wherein said emission filter will allow only a narrow range of wavelengths of light to pass and in which box is further mounted detection means for the collection and quantitation of the amount of light which passes through said rotatable polarizing means from said sample chamber to said detecting means;

wherein each of said polarizing means is a film polarizer.

21. The apparatus of claim 20, wherein said illumination means is a laser and each of said excitation lens, excitation filter and said fixed polarization means are not present.

22. A kit for the clinical diagnosis of the amount and function of a specific DNA-binding protein, which comprises a fluorescently labelled oligonucleotide and a container, wherein said oligonucleotide has the sequence of SEQ. I.D. NO. 3.

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

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

ii) contacting said labelled polynucleotide with said macromolecule, under conditions such that the concentration of said labelled polynucleotide is less than 10 nanomolar;

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

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

v) detecting formation of said complex by observing an increase in the polarization measured in step (iii) compared to the polarization measured in step (i);

wherein said polarization measurements are made using an apparatus comprising a high intensity light source as an illumination means coupled by a fiber optic cable to a sample chamber containing said polynucleotide and said macromolecule and at least one film polarizer as a polarization means.

28. The method of claim 24, wherein said fluorescent label is coumarin, eosin, fluorescein or a dansyl derivative.

29. The method of claim 24, wherein said high intensity light source is a laser or a xenon arc lamp.