LABELING AND DETECTION OF NUCLEIC ACIDS

• US 20100261181A1
• Filed: 03/23/2010
• Published: 10/14/2010
• Est. Priority Date: 02/10/2006
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Agnew, Brian, Ford, Maura J., Kumar, Kapil, Gee, Kyle R.
• Application Number:
  US12/729,903
• Publication Number:
  US 20100261181A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 207/40 2,5-Pyrrolidine-diones
• CPC Class Codes: C07D 263/32 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 295/22 with hetero atoms directly attached to ring nitrogen atoms
• CPC Class Codes: C07D 307/58 One oxygen atom, e.g. butenolide
• CPC Class Codes: C07D 311/88 Nitrogen atoms
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 2333/91245 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Provided in certain embodiments are new methods for forming azido modified nucleic acid conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling nucleic acids with an azide group.

Claims

2. A method of forming a nucleic acid conjugate, wherein the method comprises:

a) incorporating a terminal alkyne modified nucleotide into the nucleic acid polymer by contacing the terminal alkyne modified nucleotide nucleotide with at least one other nucleotide in the presence of a DNA amplification enzyme to form a terminal alkyne modified nucleic acid polymer; and

b) contacting the terminal alkyne modified nucleic acid polymer with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form a nucleic acid polymer-reporter molecule, carrier molecule, solid support conjugate.

3. A method of forming a nucleic acid conjugate, wherein the method comprises:

a) incorporating a phosphine modified nucleotide into the nucleic acid polymer by contacing the phosphine modified nucleotide nucleotide with at least one other nucleotide in the presence of a DNA amplification enzyme to form a phosphine modified nucleic acid polymer; and

b) contacting the phosphine modified nucleic acid polymer with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form a nucleic acid polymer-reporter molecule, carrier molecule, solid support conjugate.

4. A method for making an azido, alkyne or phosphine modified nucleic acid polymer, wherein the method comprises:

incubating at least one azido, alkyne or phosphine modified nucleotide in the presence of a nucleic acid amplification enzyme to form an azido, alkyne or phosphine modified nucleic acid polymer.

5. The method according to claim 4, wherein the nucleic acid enzyme is a DNA polymerase.

6. The method according to claim 4, wherein the nucleic acid enzyme is a RNA polymerase.

7. The method according to claim 4, wherein the melting temperature of the azido, alkyne or phosphine modified nucleic acid polymer is increased.

14. A method of detecting an azido modified nucleic acid polymer, comprising:

a) forming an azide-alkyne cycloaddition reaction mixture comprising;

a reporter molecule that comprises a terminal alkyne moiety;

an azido modified nucleic acid polymer;

b) incubating the azide-alkyne cycloaddition reaction mixture for a sufficient amount of time to form a nucleic acid polymer-reporter molecule conjugate;

c) separating the nucleic acid polymer-reporter conjugate by size and/or weight of the nucleic acid polymer-reporter-reporter molecule conjugate to form a separated nucleic acid polymer-reporter-reporter molecule conjugate;

d) illuminating the separated nucleic acid polymer-reporter-reporter molecule conjugate with an appropriate wavelength to form an illuminated nucleic acid polymer-reporter- reporter molecule conjugate;

e) observing the illuminated nucleic acid polymer-reporter-reporter molecule conjugate wherein the nucleic acid polymer is detected.

1. A method of forming a nucleic acid conjugate, wherein the method comprises:

a) incorporating an azide modified nucleotide into the nucleic acid polymer by contacing the azide modified nucleotide nucleotide with at least one other nucleotide in the presence of a DNA amplification enzyme to form an azide modified nucleic acid polymer; and

b) contacting the azide modified nucleic acid polymer with a reporter molecule, carrier molecule or solid support that comprises an activated or terminal alkyne or phosphine moiety to form a nucleic acid polymer-reporter molecule, carrier molecule, solid support conjugate.

8. The method according to claim 1, wherein the reporter molecule is a xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye.

9. The method according to claim 1, wherein the reporter molecule is an enzyme substrate or hapten.

10. The method according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

11. The method according to claim 1, wherein the carrier molecule comprises an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

12. The method according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

13. The method according to claim 1, wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

16. The method according to claim 14, wherein step a) further comprises a. copper ions;

b. at least one reducing agent; and

c. a copper chelator.

17. The method according to claim 14, wherein the reporter molecule is an enzyme substrate, fluorescent protein or hapten.

23. The method according to claim 14, wherein the separating step comprises chromatography or electrophoresis.

24. The method according to claim 23, wherein the chromatography comprises one or more of FPLC, HPLC, liquid chromatograpy (LC), size exclusion chromatography, ion exchange chromatography, or affinity chromatography.

25. The method according to claim 23, wherein electrophoresis comprises gel electrophoresis, 1 dimensional (1D) gel electrophoresis, 2 dimensional (2D) gel electrophoresis, native gel electrophoresis, denaturing gel electrophoresis, isoelctric focusing, or capillary electrophoresis.

16-1. The method according to claim 14, wherein the reporter molecule is xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye.

18. The method according to claim 15, wherein the copper chelator is a copper (I)chelator.

19. The method according to claim 15, wherein the copper chelator is N,N,N′

,N′

-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA, neocuproine, N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC), 1,10 phenanthroline, or a derivative thereof, trientine, glutathione, histadine, polyhistadine or tetra- ehylenepolyamine (TEPA).

20. The method according to claim 15, wherein the copper chelator is 1,10 phenanthroline, bathophenanthroline disulfonic acid (4,7odiphenyl-1,10-phenanthroline disulfonic acid) or bathocuproine disulfonic acid (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).

21. The method according to claim 15, wherein the reducing agent is acorbate, Tris(2-Carboxyethyl) Phosphine (TCEP), TCP (2,4,6-trichlorophenol), NADH, NADPH, thiosulfate, 2-mercaptoethanol, dithiothreotol, glutathione, cysteine, metallic copper, quinone, hydroquinone, vitamin K₁, Fe²⁺, Co²⁺, or an applied electric potential.

22. The method according to claim 15, wherein the reducing agent is ascorbate.

26. An azide-alkyne cycloaddition reaction mixture comprising:

a reporter molecule that comprises a terminal alkyne moiety;

an azido modified nucleic acid;

copper ions;

at least one reducing agent; and

a copper chelator.

27. A method for detecting immobilized azido modified nucleic acids, wherein the method comprises:

a) immobilizing the azido modified nucleic acids on a solid or semi-solid matrix to form an immbolized azido modified nucleic acid;

b) contacting the immbolized azido modified nucleic acid with a reporter molecule that contains an azide reactive group to form a contacted azido modified nucleic acid;

c) incubating the contacted azido modified nucleic acid for a sufficient amount of time to form a reporter molecule-nucleic acid conjugate;

d) illuminating the reporter molecule-nucleic acid conjugate with an appropriate wavelength to form an illuminated reporter molecule-nucleic acid conjugate;

e) observing the illuminated reporter molecule-nucleic acid conjugate whereby the immobilized azido modified nucleic acid is detected.

28. A method for detecting immobilized alkyne modified nucleic acids, wherein the method comprises:

a) immobilizing the alkyne modified nucleic acids on a solid or semi-solid matrix to form an immbolized alkyne modified nucleic acid;

f) contacting the immbolized alkyne modified nucleic acid with a reporter molecule that contains an azido group to form a contacted alkyne modified nucleic acid;

g) incubating the contacted alkyne modified nucleic acid for a sufficient amount of time to form a reporter molecule-nucleic acid conjugate;

h) illuminating the reporter molecule-nucleic acid conjugate with an appropriate wavelength to form an illuminated reporter molecule-nucleic acid conjugate;

i) observing the illuminated reporter molecule-nucleic acid conjugate whereby the immobilized alkyne modified nucleic acid is detected.

29. The method according to claim 28, wherein the solid or semi-solid support is a slide, an array, an agarose gel, a polyacrylamide gel, a hydrogel, a polymeric particle or glass.

30. A kit comprising:

Azide or alkyne-dUTP;

a telomerase enzyme;

an azide or alkyne reactive reporter molecule, carrier molecule or solid support.

31. A kit for labeling a nucleic acid polymer comprising:

at least one nucleotide analogue that comprises an azide, alkyne or phosphine moiety; and

a reporter molecule, carrier molecule or solid support comprising an azide, alkyne or phosphine moiety.

32. The kit according to claim 31, further comprising a nucleici acid amplification enzyme.

33. A method of measuring Telomerase Enzyme Activity, comprising steps of:

a) contacting a cell with an effective amount of a dNTP nucleotide that comprises an azide group and a Telomerase enzyme such that the dNTP nucleotide is incorporated into at least one nucleic acid polymer;

b) contacting the nucleic acid polymer with a reporter molecule comprising an alkyne or phosphine moiety to form a azido modified nucleic acid polymer reporter molecule conjugate;

c) separating the azido modified nucleic acid polymer reporter molecule conjugate from nucleic acid polymers that do not comprise a reporter molecule, and

d) illuminating the azido modified nucleic acid polymer reporter molecule conjugate to determine Telomerase activity.

34. A method of measuring Telomerase Enzyme Activity, comprising steps of:

a) contacting a cell with an effective amount of a dNTP nucleotide that comprises an alkyne or phosphine group and a Telomerase enzyme such that the dNTP nucleotide is incorporated into at least one nucleic acid polymer;

b) contacting the nucleic acid polymer with a reporter molecule comprising an azido moiety to form an alkyne or phosphine modified nucleic acid polymer reporter molecule conjugate;

c) separating the alkyne or phosphine modified nucleic acid polymer reporter molecule conjugate from nucleic acid polymers that do not comprise a reporter molecule, and d) illuminating the alkyne or phosphine modified nucleic acid polymer reporter molecule conjugate to determine Telomerase activity.