Compositions, methods, and kits for isolating nucleic acids using surfactants and proteases

• US 7,001,724 B1
• Filed: 11/28/2000
• Issued: 02/21/2006
• Est. Priority Date: 11/28/2000
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Greenfield, I. Lawrence
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/724,613
• Time in Patent Office: 1,911 Days
• Field of Search: 435/6, 435/243, 536/23.1, 536/26.42, 536/27.12
• US Class Current: 435/270
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers

Abstract

The invention provides compositions and methods for releasing and for isolating nucleic acids from biological samples, preferably from whole tissue, using cationic surfactants and proteases. The surfactant-protease combinations, when used with whole tissue, macerate the tissue, lyse individual cells, release nucleic acids, and inactivate nucleases. Kits for isolating nucleic acids from biological samples, particularly from whole tissue, are also provided.

Claims

1. A composition for macerating whole tissue, wherein the whole tissue is not a microorganism, a virus, or blood, comprising:

at least one cationic surfactant, at least one protease, a buffer, and a salt at a concentration of about 550 mM or less, wherein the cationic surfactant accelerates maceration of the whole tissue by the at least one protease.

2. The composition of claim 1, wherein the at least one cationic surfactant is protonated under the conditions used.

3. The composition of claim 1, wherein the at least one cationic surfactant has the structure:

wherein R₁, R₂, R₃, and R₄ are independently selected from the group consisting of;

—

H;

an alkyl group comprising between one and twenty carbon atoms; and

an aryl group comprising between six to twenty-six carbon atoms.

4. The composition of claim 3, wherein the cationic surfactant is an alkyltrimethyl ammonium salt, where R₁, R₂, and R₃ are methyl groups, and R₄ is an alkyl group comprising 6, 8, 10, 12, 14, 16, or 18 carbon atoms.

5. The composition of claim 4, where the cation of the alkyltrimethyl ammonium salt is selected from the group consisting of cetyltrimethylammonium, hexadecyltrimethylammonium, tetradecyltrimethylammonium, dodecyltrimethylammonium, and lauryl trimethylammonium.

6. The composition of claim 4, where the anion (X⁻

) of the alkyltrimethyl ammonium salt is bromide, chloride, iodide, hydroxide, nitrate, sulfate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, or citrate.

7. The composition of claim 3, wherein the at least one cationic surfactant is a benzyldimethyl-n-alkylammonium salt, where R₁ and R₂ are methyl groups, R₃ is an aryl group comprising six carbon atoms, and R₄ is an alkyl group comprising 6, 8, 10, 12, 14, 16, or 18 carbon atoms.

8. The composition of claim 7, where the anion of the benzyldimethyl-n-alkylammonium salt is selected from the group consisting of bromide, chloride, iodide, hydroxide, nitrate, sulfate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, and citrate.

9. The composition of claim 1, wherein the at least one protease is selected from the group consisting of subtilisins, subtilases, and alkaline serine proteases.

10. The composition of claim 1, wherein the at least one protease is selected from the group consisting of Proteinase K, Proteinase R, Proteinase T, Subtilisin DY, an alkaline serine protease from Streptomyces griseus or Bacillus licheniformis, Dispase, subtilisin Carlsberg, subtilopeptidase A, and thermolysin.

11. The composition of claim 9, wherein the protease is Proteinase K.

12. The composition of claim 9, wherein the protease is thermolysin or a thermostable protease.

13. The composition of claim 12, wherein the protease is from Thermus Rt41A or Bacillus thermoproteolyticus rokko.

14. The composition of claim 1, further comprising calcium chloride.

15. The composition of claim 1, wherein the buffer maintains the pH between pH 7 and pH 9.

16. The composition of claim 1, wherein the buffer maintains the pH between pH 5 and pH 7.

17. The composition of claim 1, further comprising at least one ribonuclease inhibitor.

18. The composition of claim 17, wherein the at least one ribonuclease inhibitor comprises aurintricarboxylic acid, vanadylate ribonucleoside complexes, phenylglyoxal, p-hydroxyphenylglyoxal, polyamines, spermidine, 9-aminoacridine, iodoacetate, bentonite, poly[2′

-O-(2,4-dinitrophenyl)]poly(adenyhlic acid), zinc sulfate, bromopyruvic acid, formamide, copper, or zinc.

19. The composition of claim 18, wherein the at least one ribonuclease inhibitor comprises aurintricarboxylic acid.

20. The composition of claim 1, wherein the cationic surfactant is cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTACI), hexadecyltrimethylammonium bromide, or hexadecyltrimethylammonium chloride;

the protease is Proteinase K;

the buffer maintains the pH between pH 5 and pH 7; and

further comprising aurintricarboxylic acid.

21. The composition of claim 20, further comprising at least one solubilizing agent for enhancing the solubility or permeability of the sample.

22. The composition of claim 21, wherein the solubilizing agent is 1-methyl 2 pyrolidinone, N-methylpyrolidinone, pyrolidinone, dimethylformamide, or dimethylsulfoxide.

23. The composition of claim 1, further comprising at least one deoxyribonuclease inhibitor.

24. The composition of claim 23, wherein the at least one deoxyribonuclease inhibitor comprises a divalent cation chelator.

25. The composition of claim 24, wherein the chelator is EDTA, EGTA, of DPTA.

26. A composition for macerating whole tissue, wherein the whole tissue is not a microorganism, a virus, or blood, comprising:

at least one cationic surfactant, at least one protease, a buffer, and the whole tissue, wherein the at least one cationic surfactant is selected from;

a) an alkyltrimethyl ammonium salt having the structure;

wherein R₁, R₂, and R₃ are methyl groups, and R₄ is an alkyl group, comprising 6, 8, 10, 12, 14, 16, or 18 carbon atoms; and

b) a benzyldimethyl-n-alkylammonium salt having the structure;

wherein R₁ and R₂ are methyl groups. R₃ is an aryl group comprising six carbon atoms, and R₄ is an alkyl group comprising 6, 8, 10, 12, 14, 16, or 18 carbon atoms.

27. The composition of claim 26, wherein the at least one cationic surfactant is protonated under the conditions used.

28. The composition of claim 26, wherein the at least one cationic surfactant is the alkyltrimethyl ammonium salt of a), and where the cation of the alkyltrimethyl ammonium salt is selected from the group consisting of cetyltrimethylammonium, hexadecyltrimethylammonium, tetradecyltrimethylammonium, dodecyltrimethylammonium, and lauryl trimethylammonium.

29. The composition of claim 26, wherein the at least one cationic surfactant is the alkyltrimethyl ammonium salt of a), and where the anion (X⁻

) of the alkyltrimethyl ammonium salt is bromide, chloride, iodide, hydroxide, nitrate, sulfate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, or citrate.

30. The composition of claim 26, wherein the at least one cationic surfactant is the benzyldimethyl-n-alkylammonium salt of b), and where the anion of the benzyldimethyl-n-alkylammonium salt is selected from the group consisting of bromide, chloride, iodide, hydroxide, nitrate, sulfate, phosphate, formate, acetate, propionate, oxalate, malonate, succinate, and citrate.

31. The composition of claim 26, wherein the at least one protease is selected from the group consisting of subtilisins, subtilases, and alkaline serine proteases.

32. The composition of claim 31, wherein the at least one protease is selected from the group consisting of Proteinase K, Proteinase R, Proteinase T, Subtilisin DY, an alkaline serine protease from Streptomyces griseus or Bacillus licheniformis, Dispase, subtilisin Carlsberg, subtilopeptidase A, and thermolysin.

33. The composition of claim 31, wherein the protease is Proteinase K.

34. The composition of claim 31, wherein the protease is thermolysin or a thermostable protease.

35. The composition of claim 34, wherein the protease is from Thermus Rt41A or Bacillus thermoproteolyticus rokko.

36. The composition of claim 26, further comprising calcium chloride.

37. The composition of claim 26, wherein the buffer maintains the pH between pH 7 and pH 9.

38. The composition of claim 26, wherein the buffer maintains the pH between pH 5 and pH 7.

39. The composition of claim 26, further comprising at least one ribonuclease inhibitor.

40. The composition of claim 39, wherein the at least one ribonuclease inhibitor comprises aurintricarboxylic acid, vanadylate ribonucleoside complexes, phenylglyoxal, p-hydroxyphenylglyoxal, polyamines, spermidine, 9-aminoacridine, iodoacetate, bentonite, poly[2′

-O-(2,4-dinitrophenyl)]poly(adenyhlic acid), zinc sulfate, bromopyruvic acid, formamide, copper, or zinc.

41. The composition of claim 39, wherein the at least one ribonuclease inhibitor comprises aurintricarboxylic acid.

42. The composition of claim 26, wherein the cationic surfactant is cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTACI), hexadecyltrimethylammonium bromide, or hexadecyltrimethylammonium chloride;

the protease is Proteinase K;

the buffer maintains the pH between pH 5 and pH 7; and

further comprising aurintricarboxylic acid.

43. The composition of claim 42, further comprising at least one solubilizing agent for enhancing the solubility or permeability of the sample.

44. The composition of claim 43, wherein the solubilizing agent is 1-methyl 2 pyrolidinone, N-methylpyrolidinone, pyrolidinone, dimethylformamide, or dimethylsulfoxide.

45. The composition of claim 26, further comprising at least one deoxyribonuclease inhibitor.

46. The composition of claim 45, wherein the at least one deoxyribonuclease inhibitor comprises a divalent cation chelator.

47. The composition of claim 46, wherein the chelator is EDTA, EGTA, of DPTA.