Methods of using dyes in association with nucleic acid staining or detection and associated technology

• US 20060211029A1
• Filed: 03/16/2006
• Published: 09/21/2006
• Est. Priority Date: 03/17/2005
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Leung, Wai-Yee, Mao, Fei
• Granted Patent: US 7,601,498 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C09B 11/28 Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods of using dyes and associated technology are provided. A dye, such as a monomeric dye or a dimeric dye, may be used in a nucleic acid gel staining application and/or a nucleic acid detection application. Such a dye and a salt that comprises an anion that is associated with a strong acid and a cation that is associated with a strong base may be used in such an application. A dimeric dye, such as a dimeric dye capable of forming a hairpin-like structure, may be used to stain and/or detect nucleic acids via a release-on-demand mechanism. A dimeric dye having low background fluorescence in the absence of nucleic acids and high fluorescence in the presence of nucleic acids, upon binding therewith, may be used to stain and/or detect nucleic acids.

Claims

1. A method of determining presence or absence of nucleic acid in a sample, wherein if nucleic acid is present it is immobilized relative to a matrix or a surface, the method comprising:

exposing the nucleic acid to a fluorescent nucleic acid dye having the formula;

wherein BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 8 to about 150 non-hydrogen atoms, inclusive;

Q₁ is a fluorescent nucleic acid dye constituent;

Q₂ is a fluorescent nucleic acid dye constituent; and

Q₁ and Q₂ may be the same or different;

such that, if nucleic acid is present in the sample, a complex of the fluorescent nucleic acid dye and the nucleic acid is formed; and

detecting fluorescence associated with the complex or a lack thereof.

2. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent is selected from an acridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye, a pyronin dye and a styryl dye.

3. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

4. The method of claim 3, wherein each R₁ is H;

R₂ is H;

R₃ represents where BRIDGE attaches to the structure;

each R₆ is —

CH₃; and

each R₇ is —

CH₃.

5. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

6. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

7. The method of claim 1, wherein at least one dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

8. The method of claim 1, wherein BRIDGE has the formula:

-L-[A¹-(CH₂)_α

-]_a[A²-(CH₂)_β

-]_b[A³-(CH₂)_γ

-]_c[A⁴-(CH₂)_δ

-]_d[A⁵-(CH₂)_ε

-]_e[A⁶-(CH₂)_ζ

-]_f[A⁷-(CH₂)_η

-]_g[A⁸-(CH₂)_θ

-]_h[A⁹-(CH₂)_ι

-]_i-A¹⁰-L-

wherein each L, independently, is a moiety comprising a single bond;

a polymethylene unit having 1 carbon to about 12 carbons, inclusive, optionally comprising at least one hetero atom selected from N, O and S;

or an aryl optionally comprising at least one hetero atom selected from N, O and S;

each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is a nucleic-acid-binding-enhancing-group (NABEG);

a branched alkyl optionally comprising at least one hetero atom selected from N, O and S;

or at least one saturated 5- or 6-membered ring, optionally comprising at least one hetero atom selected from N, O and S;

each of α

, β

, γ

, δ

, ε

, ζ

, η

, θ

, and ι

, independently, is zero or an integer from 1 to about 20, inclusive; and

each of a, b, c, d, e, f, g, h, and i, independently, is zero or an integer from 1 to about 20, inclusive.

9. The method of claim 8, wherein BRIDGE comprises from about 10 to about 100 non-hydrogen atoms, inclusive.

10. The method of claim 8, wherein each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is a NABEG comprising a moiety that comprises at least one bond linkage that comprises at least one amide bond, urethane bond, urea bond, thiourea bond, ether bond, or thioether bond;

or an aryl optionally comprising at least one hetero atom selected from halogens, N, O, and S.

11. The method of claim 1, wherein BRIDGE has the formula:

—

(CH₂)_x—

C(═

O)NH—

(CH₂)_α

—

[O—

(CH₂)_β

]_b—

[O—

(CH₂)_γ

]_c—

NH(O═

C)—

(CH₂)_x—

wherein each x, independently, is an integer selected from 1 to 11, inclusive;

α

is an integer selected from 2 to about 20, inclusive;

each of β and

γ

, independently, is 2 or 3;

b is zero or an integer from 1 to about 20, inclusive; and

c is zero or 1.

12. The method of claim 11, wherein x is 5;

α and

γ

are the same and are 2 or 3;

β

is 2;

b is 0, 1, 2, or 3; and

c is 1.

13. The method of claim 11, wherein Q₁ and Q₂ are the same.

14. The method of claim 11, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

15. The method of claim 11, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

16. The method of claim 11, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

17. The method of claim 11, wherein each dye constituent of the Q₁ dye constituent and the Q₂ dye constituent has the structure:

18. The method of claim 1, wherein the fluorescent nucleic acid dye has the structure:

19. The method of claim 1, wherein the fluorescent nucleic acid dye has the structure:

20. The method of claim 1, wherein the fluorescent nucleic acid dye has the structure:

21. The method of claim 1, wherein if nucleic acid is present in the sample, nucleic acid is immobilized relative to a solid matrix, a semi-solid matrix, or a solid surface.

22. The method of claim 1, wherein if nucleic acid is present in the sample, nucleic acid is immobilized relative to a membrane surface, a glass surface, a plastic surface, or a polysilicon surface.

23. The method of claim 1, wherein if nucleic acid is present in the sample, nucleic acid is immobilized relative to a solid matrix or a semi-solid matrix.

24. The method of any of claim 1, claim 11, claim 18, claim 19, and claim 20, wherein if nucleic acid is present in the sample, nucleic acid is immobilized relative to a gel matrix.

25. The method of claim 24, wherein the gel matrix comprises agarose or polyacrylamide.

26. The method of claim 24, wherein said exposing comprises pre-cast gel staining.

27. The method of claim 24, wherein said exposing comprises post-gel staining.

28. A method of staining a sample that is exposed to a matrix or a surface, wherein if nucleic acid is present in the sample it is capable of being immobilized relative to the matrix or the surface, comprising:

providing an aqueous solution comprising a fluorescent nucleic acid dye and a salt that comprises an anion that would be sufficient as a component of a strong acid and a cation that would be sufficient as a component of a strong base; and

exposing the sample to the aqueous solution.

29. The method of claim 28, wherein a concentration of the salt is from about 5 mM to about 0.5 M, inclusive.

30. The method of claim 28, wherein a concentration of the salt is about 0.05 M to about 0.2 M, inclusive.

31. The method of claim 28, wherein the strong acid has a pKa of about 2 or less and the strong base has a pKa of about 10 or more.

32. The method of claim 28, wherein the salt is selected from NaCl, sodium bromide, sodium sulfate, potassium chloride, potassium bromide, potassium sulfate, magnesium choride and tetramethylammonium chloride.

33. The method of claim 28, wherein the fluorescent nucleic acid dye is a monomeric dye or a dimeric dye.

34. A kit for determining presence or absence of nucleic acid in a sample, wherein if nucleic acid is present in the sample it is capable of being immobilized relative to the matrix or the surface, the kit comprising:

a fluorescent nucleic acid dye having the structure;

wherein BRIDGE is a substantially aliphatic, substantially neutral linker comprising from about 8 to about 150 non-hydrogen atoms, inclusive;

Q₁ is a fluorescent nucleic acid dye constituent;

Q₂ is a fluorescent nucleic acid dye constituent; and

Q₁ and Q₂ may be the same or different;

optionally, a buffer;

optionally, a gel matrix, at least one material for forming a gel matrix, a surface, or at least one material for forming a surface; and

information concerning use of the fluorescent nucleic acid dye.

35. The kit of claim 34, wherein the fluorescent nucleic acid dye is in an aqueous solution.

36. The kit of claim 34, wherein the fluorescent nucleic acid dye is in a gel matrix.

37. The kit of claim 34 or 36, wherein the gel matrix comprises agarose.