Methods of using dyes in association with nucleic acid staining or detection and associated technology
First Claim
1. A method of determining presence or absence of nucleic acid in a sample, the method comprising:
- exposing the nucleic acid to a fluorescent nucleic acid dye having the formula;
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Accused Products
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.
130 Citations
37 Claims
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1. A method of determining presence or absence of nucleic acid in a sample, the method comprising:
exposing the nucleic acid to a fluorescent nucleic acid dye having the formula;
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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.
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3. The method of claim 1, wherein each R1 of Formula III is H;
- R2 of Formula III is H;
R3 of Formula III represents where BRIDGE attaches to the structure;
each R6 of Formula III is —
CH3; and
each R7 of Formula III is —
CH3.
- R2 of Formula III is H;
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4. The method of claim 1, wherein one dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure:
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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:
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6. 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; andeach of a, b, c, d, e, f, g, h, and i, independently, is zero or an integer from 1 to about 20, inclusive.
-
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7. The method of claim 6, wherein BRIDGE comprises from about 10 to about 100 non-hydrogen atoms, inclusive.
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8. The method of claim 6, 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.
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9. 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.
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10. The method of claim 9, 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.
- α and
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11. The method of claim 9, wherein Q1 and Q2 are the same.
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12. The method of claim 9, wherein each dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure:
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13. The method of claim 9, wherein each dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure of Formula II:
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14. The method of claim 9, wherein each dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure:
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15. The method of claim 9, wherein each dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure:
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16. The method of claim 1, wherein the fluorescent nucleic acid dye has the structure:
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17. The method of claim 1, wherein the fluorescent nucleic acid dye has the structure:
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18. The method of claim 1, wherein the fluorescent nucleic acid dye has the structure:
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19. A method of determining presence or absence of nucleic acid in a sample, the method comprising:
exposing the nucleic acid to a fluorescent nucleic acid dye having the formula;
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20. The method of claim 19, wherein -L-BRIDGE-L- has the formula:
-
—
(CH2)x—
C(═
O)NH—
(CH2)α
—
[O—
(CH2)β
]b—
[O—
(CH2)γ
]c—
NH(O═
C)—
(CH2)x—where in each x, independently, is an integer selected from 1 to 11, inclusive;
a 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.
-
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21. The method of claim 1 or 19, 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.
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22. The method of claim 1 or 19, 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.
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23. The method of claim 1 or 19, wherein if nucleic acid is present in the sample, nucleic acid is immobilized relative to a solid matrix or a semi-solid matrix.
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24. The method of any of claim 1, claim 9, claim 16, claim 17, 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.
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25. The method of claim 24, wherein the gel matrix comprises agarose or polyacrylamide.
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26. The method of claim 24, wherein said exposing comprises pre-cast gel staining.
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27. The method of claim 24, wherein said exposing comprises post-gel staining.
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28. The method of claim 19, wherein at least one dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure:
-
29. The method of claim 28, wherein each R1 is H;
- R2 is H;
R3 represents where an L of -L-BRIDGE-L- attaches to the structure;
each R6 is —
CH3; and
each R7 is —
CH3.
- R2 is H;
-
30. The method of claim 19, wherein at least one dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure;
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31. The method of claim 19, wherein BRIDGE has the formula:
-
-[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-wherein 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; andeach of a, b, c, d, e, f, g, h, and i, independently, is zero or an integer from 1 to about 20, inclusive.
-
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32. The method of claim 19, wherein -L-BRIDGE-L- comprises from about 10 to about 100 non-hydrogen atoms, inclusive.
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33. The method of claim 31, wherein each of 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.
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34. The method of claim 20, 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.
- α and
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35. The method of claim 20, wherein Q1 , and Q2 are the same.
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36. The method of claim 20, wherein each dye constituent of the Q1 , dye constituent and the Q2 dye constituent has the structure:
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37. The method of claim 20, wherein each dye constituent of the Q1 dye constituent and the Q2 dye constituent has the structure:
Specification