Hybridization compositions and methods
The invention provides methods and compositions for hybridizing at least one molecule to a target. The invention may, for example, eliminate the use of or reduce the dependence on formamide in hybridization. Compositions for use in the invention include an aqueous composition comprising at least one nucleic acid sequence and at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences.
1. A hybridization composition comprising a mixture of at least one nucleic acid sequence, at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences, an accelerating agent, and at least one additional component selected from the group consisting of: buffering agents, salts, chelating agents, detergents, and blocking agents, wherein less than 10% of the hybridization composition is formamide, wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO) and has lactone, sulfone, sulfite, and/or carbonate functional groups and wherein the at least one nucleic acid sequence is a FISH or CISH probe;
wherein the at least one polar aprotic solvent comprises a polar aprotic solvent selected from the group consisting of:
and
wherein the accelerating agent is selected from (i) a polymer, (ii) a protein, (iii) a glycol, (iv) glycerol, (v) combinations thereof, and (vi) organic solvents.
2. The hybridization composition according to claim 1 , wherein the concentration of polar aprotic solvent is about 1% to 90% (v/v).
3. The hybridization composition according to claim 1 or 2 , wherein the concentration of polar aprotic solvent is 5% to 10% (v/v).
4. The hybridization composition according to claim 1 or 2 , wherein the concentration of polar aprotic solvent is 10% to 20% (v/v).
5. The hybridization composition according to claim 1 or 2 , wherein the concentration of polar aprotic solvent is 20% to 30% (v/v).
6. The hybridization composition according to claim 1 , wherein the polar aprotic solvent is non-toxic.
7. The hybridization composition according to claim 1 , with the proviso that the composition does not contain formamide.
8. The hybridization composition according to claim 1 , wherein the composition contains less than 2% formamide.
9. The hybridization composition according to claim 1 wherein the composition contains less than 1% formamide.
10. The hybridization composition according to claim 1 , wherein the polar aprotic solvent has a dispersion solubility parameter between 17.7 to 22.0 MPa1/2, a polar solubility parameter between 13 to 23 MPa1/2, and a hydrogen bonding solubility parameter between 3 to 13 MPa1/2.
11. The hybridization composition according to claim 1 , wherein the further at least one polar aprotic solvent is selected from the group consisting of: 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, dimethyl sulfate, dimethyl sulfone, diphenyl sulfone, ethanesulfonylchloride, ethylene carbonate, ethylene glycol sulfate, glycol sulfite, methyl alpha bromo tetronate, methyl phenyl sulfone, methyl sulfolane, methyl-4-toluenesulfonate, N-phenyl sydnone, phthalic anhydride, 1,3-propane sultone, β-propiolactone, propylene carbonate, saccharin, sulfanilamide, sulfolane, trimethylene sulfide-dioxide, and trimethylene sulfite.
12. The hybridization composition according to claim 1 , wherein the salt is NaCl and/or the buffering agent is phosphate buffer.
13. The hybridization composition according to claim 12 , wherein the composition further comprises dextran sulfate present at a concentration of 10% to 40%, and wherein the NaCl is present at a concentration of 0 mM to 1200 mM, and/or the phosphate buffer is present at a concentration of 0 mM to 50 mM.
14. The hybridization composition according to claim 13 , wherein the dextran sulfate is present at a concentration of 10% to 30%, the NaCl is present at a concentration of 300 mM to 600 mM, and/or the phosphate buffer is present at a concentration of 5 mM to 20 mM.
15. The hybridization composition according to claim 1 , wherein:
the accelerating agent is selected from the group consisting of: formamide, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer.
16. The hybridization composition according to claim 1 , wherein:
the accelerating agent is selected from the group consisting of: formamide at a concentration of 0.1-5%, glycerol at a concentration of 0.1% to 10%, propylene glycol at a concentration of 0.1% to 10%, 1,2-propanediol at a concentration of 0.1% to 10%, diethylene glycol at a concentration of 0.1% to 10%, ethylene glycol at a concentration of 0.1% to 10%, glycol at a concentration of 0.1% to 10%, and 1,3 propanediol at a concentration of 0.1% to 10%, and
the buffer is citric acid buffer at a concentration of 1 mM to 50 mM.
17. The hybridization composition according to claim 1 , wherein the blocking agent is selected from the group consisting of: total human DNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA.
18. The hybridization composition according to claim 17 , wherein the total human DNA, herring sperm DNA, salmon sperm DNA, or calf thymus DNA is present at a concentration of 0.01 to 10 μg/μL.
19. The hybridization composition according to claim 1 , comprising 40% of at least one polar aprotic solvent, 300 mM NaCl, and 5 mM phosphate buffer.
20. The hybridization composition according to claim 1 , comprising 15% (v/v) of at least one polar aprotic solvent, 600 mM NaCl, 10 mM phosphate buffer, 0.1 μg/μL total human DNA and further comprising 20% dextran sulfate.
21. The hybridization composition according to claim 1 , comprising:
at least one polar aprotic solvent at a concentration of 15% (v/v),
600 mM NaCl, 10 mM citric acid buffer pH 6.2,
0.1 μg/μL herring sperm DNA, salmon sperm DNA, or calf thymus DNA, or
0.5% formamide, 1% ethylene glycol, or 1% 1,3 propanediol and further comprising 20% dextran sulfate.
22. The hybridization composition according to claim 1 , comprising more than one phase at room temperature.
23. The hybridization composition according to claim 22 , comprising two phases at room temperature.
24. The hybridization composition according to claim 22 , comprising three phases at room temperature.
25. The hybridization composition according to claim 1 , wherein the at least one nucleic acid sequence is a FISH DNA probe.
26. The hybridization composition according to claim 1 , wherein the at least one nucleic acid sequence comprises a detectable label.
27. The hybridization composition according to claim 1 , further comprising up to 10% dextran sulfate.
28. The hybridization composition according to claim 1 , further comprising up to 20% dextran sulfate.
29. The hybridization composition according to claim 1 , further comprising an accelerating agent at a concentration from 1%-80%.
30. The hybridization composition according to claim 1 , wherein the accelerating agent is dextran sulfate.
31. The hybridization composition according to claim 1 , wherein the accelerating agent is selected from the group consisting of PVP, heparin, dextran sulfate, BSA, ethylene glycol, 1,3 propanediol, propylene glycol, diethylene glycol, glycerol, combinations thereof, formamide, dimethylformamide and DMSO.