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

• US 9,102,835 B2
• Filed: 08/08/2013
• Issued: 08/11/2015
• Est. Priority Date: 03/17/2005
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Mao, Fei, Leung, Wai-Yee
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/962,668
• Publication Number:
  US 20140073058A1
• Time in Patent Office: 733 Days
• Field of Search: 546/79, 546/80, 546/88, 536/26.6
• US Class Current: 1/1
• 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 compound having the formula:

2. The compound of claim 1, wherein BRIDGE has the formula:

-L₁-[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-L₂-

wherein each of L₁ and L₂, independently, is a moiety comprising a single bond;

a polymethylene unit having 1 carbon to 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;

or an aryl group optionally comprising at least one hetero atom selected from halogens, 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 20, inclusive; and

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

3. The compound of claim 2, 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;

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

each of β and

γ

, independently, is 2 or 3;

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

c is zero or 1.

4. The compound of claim 1, wherein each of Q₁ and Q₂ is a phenanthridium dye of Formula I:

5. The compound of claim 4, wherein x is 5;

α and

γ

are the same and are 2 or 3;

β

is 2; and

b is 0, 1, 2, or 3.

6. The compound of claim 4, wherein the compound has the structure:

7. The compound of claim 1, wherein each of Q₁ and Q₂ is an asymmetric cyanine dye, and wherein each of Q₁ and Q₂ dye constituents independently has a structure of Formula II:

8. The compound of claim 7, wherein each of Q₁ and Q₂ has the structure:

9. The compound of claim 7, wherein the compound has the structure:

10. The compound of claim 1, wherein each of Q₁ and Q₂ is independently an acridine dye of Formula III:

11. The compound of claim 4, 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.

12. The compound of claim 11, wherein the compound has the structure:

13. The compound of any one of claims 1-12, wherein ψ

is I⁻

or Cl⁻

.

14. A kit for determining presence or absence of nucleic acid in a sample, the kit comprising:

a compound according to any one of claims 1-13;

optionally, a buffer; and

information concerning use of the kit.

15. The compound of claim 2, wherein the Bridge comprises a weakly basic constituent.

16. The compound of claim 15, wherein the Bridge comprises an aryl group comprising at least one Nitrogen atom.

17. The compound of claim 16, wherein the Bridge comprises a pyridine group, pyrazine group, triazole group, or tetrazole group.

18. The compound of claim 2, wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ independently comprises an aryl group;

the aryl group optionally comprising at least one hetero atom selected from halo, N, O, and S.

19. The compound of claim 18, wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ independently comprises an aryl group;

the aryl group comprising at least one Nitrogen atom.

20. The compound of claim 19, wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ independently comprises a pyridine group, a pyrazine group, a triazole group, or a tetrazole group.

21. The compound of claim 20, wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ independently comprises a triazole group.

22. The compound of claim 21, wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently comprises a 1,2,3-triazole group.