Methods of using labeled probe molecules to quantify target molecules

• US 6,811,973 B1
• Filed: 11/22/2000
• Issued: 11/02/2004
• Est. Priority Date: 11/24/1999
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Reich, Norbert
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/721,550
• Time in Patent Office: 1,441 Days
• Field of Search: 435/6, 435/283.1, 435/287.1, 435/287.2, 435/288.3, 435/288.7, 435/289.1, 435/299.1, 435/305.1, 422/50, 422/68.1, 436/501, 536/23.1, 536/24.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/22.1
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence

Abstract

An assay system using labeled probe molecules to identify and quantify target molecules in a sample is disclosed. Where labeled probe molecules are present on a substrate such as a microarray, the identification of multiple different target molecules may be examined simultaneously. Alternatively, a known number of like probe molecules may be present on a substrate and a single target molecule may be quantified in a sample. Preferred labeled probe molecules are comprised fluorescent single stranded nucleotide analogs whose fluorescence is quenched by pairing with a homologous nucleotide target sequence.

Claims

1. A method for quantifying the amount of a target molecule in solution comprising the steps of:

a. incorporating one or more fluorescing nucleotide analogs into nucleotide probes to provide fluorescing nucleotide probes;

b. providing a first substrate having a surface area;

c. affixing a known number of said fluorescing nucleotide probes onto the substrate;

d. detecting a first level of fluorescence from said fluorescing nucleotide probes on the substrate;

e. contacting said first substrate with a sample solution comprising unlabeled target nucleotide sequences;

f. providing sufficient conditions and time for unlabeled target molecules to selectively hybridize with fluorescing nucleotide probes on said substrate wherein hybridization of an unlabeled target molecule and an fluorescing nucleotide probe quenches fluorescence from said fluorescing nucleotide probe;

g. removing the first substrate and detecting a second level of fluorescence from said fluorescing nucleotide probes after hybridization;

h. repeating steps a. through g with subsequent substrates, having surface areas comprising known numbers of fluorescing nucleotide probes until all target molecules are hybridized and no longer quench said fluorescing nucleotide probes; and

i. quantifying the amount of target molecule in the sample solution by adding the known number of fluorescing nucleotidc probes present on the first substrate and subsequent substrates contacted with and quenched by the unlabeled target molecule whereby the amount of the target molecule is quantified.

2. The method of claim 1, wherein said fluorescing nucleotide probes are comprised of native and nonnative nucleotides.

3. The method of claim 1, wherein the fluorescing nucleotide analogs are nucleotide analogs, including 2-amino purine for adenosine or quanine;

ribonucleoside or 2,6-diamino ribonucleoside, formycin A, formycin B, oxyformycin B, toyocamycin, sangivamycin, pseudoouridine, showdomycin, minimycin, pyrazomycin, 5-amino-formycin A, 5-amino-formycin B or 5-oxo-formycin A for adenosine;

4-amino-pyrazolo [3,4d] pyrimidine, 4,6-diamino-pyrazolo [3,4d] pyrimidine, 4-oxo-pyrazolo [3,4d] pyrimidine;

4-oxo-6-amino-pyrazolo [3,4d] pyrimidine, 4,6-dioxo-pyrazolo [3,4d] pyrimidine, pyrazolo [3,4d] pyrimidine, 6-amino-pyrazolo [3,4d] pyrimidine or 6-oxo-pyrazolo [3,4d] pyrimidine for cytosine or thymidine.

4. The method of claim 1, wherein said one or more fluorescing nucleotide analogs fluoresces at a wavelength of about 300 nm to about 700 nm.

5. The method of claim 1, wherein said fluorescing nucleotide probes are further comprised of amino acids.

6. The method of claim 1, wherein said surface area has from about 100 to about 10,000 different fluorescing nucleotide probe molecules.

7. The method of claim 1, wherein the substrate is a bead.

8. The method of claim 7, wherein said bead size ranges from about 10 microns to about 20 microns.

9. The method of claim 7, wherein the bead is formed of a ferromagnetic metal core and a polymeric coating.

10. The method of claim 7, having from about 100 to about 1,000 labeled fluorescing nucleotide probe molecules attached to the surface area of the bead.

11. The method for quantifying the amount of a target molecule in a sample solution comprising the steps of:

a. incorporating a nucleotide analog including 2-auminopurine into nucleotide probes to provide fluorescing nucleotide probes;

b. affixing a known number of said the fluorescing probes onto a substrate;

c. detecting a first level of fluorescence from said fluorescing nucleotide probes on the substrate;

d. contacting said substrate with said sample solution containing unlabeled target molecules;

e. providing sufficient conditions and time for unlabeled target molecules in said solution to selectively pair and hybridize with said fluorescing nucleotide probes affixed on said substrate wherein hybridization of an unlabeled target molecule and fluorescing probe quenches fluorescence of the nucleotide probes;

f. removing said substrate from the solution and detecting a second level of fluorescence from the fluorescing nucleotide probes on the substrate;

g. comparing said first and second level of fluorescence;

h. repeating steps d. though g. by re-contacting said sample solution with said substrate or additional substrates having a known number of fluorescing nucleotide probes until target molecules no longer quench the fluorescence from said fluorescing probes; and

i. quantifying the amount of target molecules by determining the number of quenched fluorescing probes.

12. The method of claim 11, wherein said fluorescing nucleotide probes are comprised of native and nonnative nucleotides.

13. The method of claim 11, wherein the fluorescing nucleotide probe molecules am comprised of amino acids.

14. The method of claim 11, wherein the substrate is a bead.

15. The method of claim 14, wherein said bead size ranges from about 10 microns to about 20 microns.

16. The method of claim 14, wherein the bead is formed of a ferromagnetic metal core and a polymeric coating.

17. The method of claim 14, having from about 100 to about 1,000 fluorescing nucleotide probe molecules attached to the surface area of the bead.

18. The method of claim 14, wherein the level of label expression is evaluated using a flow cytometer.

19. The method of claim 14, wherein the second level is significantly lower than the first level and said second levels of fluorescence approach zero and/or about background levels.