Compositions and methods for recovery of nucleic acids or proteins from tissue samples fixed in cytology media

• US 9,410,146 B2
• Filed: 09/14/2010
• Issued: 08/09/2016
• Est. Priority Date: 09/14/2009
• Status: Expired due to Fees

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Liu, Shubing, Ramachandran, Suganthi
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/881,531
• Publication Number:
  US 20110065906A1
• Time in Patent Office: 2,156 Days
• Field of Search: 436/17, 435/6.1, 536/25.4
• US Class Current: 1/1
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

Abstract

The present invention provides compositions and methods for improving nucleic acid or protein recovery from fixed biological samples.

Claims

1. A method of lysing a fixed biological sample, the method comprising lysing the fixed biological sample with a composition comprising at least two amines, wherein the method is useful for both aldehyde-based fixatives and alcohol-based fixatives, and wherein the pH of the composition is 8 or greater.

2. A method of isolating a component of a fixed biological sample, the method comprising:

a. lysing the fixed biological sample according to the method of claim 1; and

b. isolating the component from the lysate.

3. The method of claim 2, wherein at least one of the amines is selected from the group consisting of methylamine, dimethylamine, diethylamine, trimethlylamine, triethylamine, monoethanolamine, diethanolamine, triethanolamine, ethylenediamine, diethylenetriamine, tris(hydroxymethyl)aminomethane, hexamethylenetetramine, aniline, and an amino acid.

4. The method of claim 2, wherein at least one of the amines is a buffering agent.

5. The method of claim 4, wherein the buffering agent is selected from the group consisting of tris(hydroxymethyl)aminomethane (“

TRIS”

), N-tris-(hydroxymethyl)methyl-3-aminopropanesulfonic acid (“

TAPS”

), 3-[N-tris-(hydroxymethyl)-methyl-amino]-2-hydroxypropanesulfonic acid (“

TAPSO”

);

N-tris(hydroxymethyl)methyl-2-am inoethanesulfonic acid (“

TES”

);

N-[tris(hydroxymethyl)methyl]-glycine (“

TRICINE”

);

bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane (“

bis-TRIS”

); and

1,3-bis[tris(hydroxy-methyl)methylamino]propane (“

bis-TRIS PROPANE”

).

6. The method of claim 4, wherein the buffering agent is TRIS.

7. The method of claim 2, wherein at least one of the amines is diazoamine.

8. The method of claim 2, wherein the composition further comprises at least one detergent.

9. The method of claim 8, wherein the detergent is selected from the group consisting of polyoxyethyleneglycol dodecyl ether, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, tert-octylphenoxy poly(oxyethylene)ethanol (NONIDET P-40), Igepal CA-630, deoxycholate, 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100), sodium dodecyl sulfate, and polysorbate surfactants.

10. The method of claim 2, wherein the composition further comprises at least one DNA-binding magnetic bead.

11. The method of claim 2, wherein the composition comprises:

150 mM Tris-HCl;

300 mM diethanolamine;

3% Brij-58;

0.09% sodium azide; and

wherein the composition has a pH of approximately 9.4.

12. The method of claim 1, wherein the fixed biological sample is fixed with a cross-linking fixative agent.

13. The method of claim 12, wherein the cross-linking fixative agent comprises an aldehyde.

14. The method of claim 13, wherein the aldehyde is selected from the group consisting of formaldehyde and glutaraldehyde.

15. The method of claim 1, wherein the fixed biological sample is fixed with a fixative agent comprising an alcohol.

16. The method of claim 15, wherein the alcohol is selected from the group consisting of methanol and ethanol.

17. The method of claim 1, wherein the fixed biological sample is fixed with a fixative agent selected from the group consisting of the aldehyde-based cytology medium SUREPATH™

, and the alcohol-based cytology medium PRESERVCYT™

.

18. The method of claim 1, wherein the fixed biological sample is a cervical sample.

19. The method of claim 2, wherein the component is a nucleic acid molecule.

20. The method of claim 19, wherein the nucleic acid molecule comprises a specific sequence.

21. The method of claim 20, wherein the nucleic acid is isolated according to a method comprising hybridizing a nucleic acid probe to the specific sequence of the nucleic acid molecule.

22. The method of claim 21, wherein the nucleic acid probe is adapted to be bound to a solid phase or is bound to a solid phase.

23. The method of claim 22, wherein the solid phase is a magnetic bead.

24. The method of claim 21, wherein the hybridizing of the nucleic acid probe to the specific sequence of the nucleic acid molecule results in the formation of a DNA:

RNA hybrid between the nucleic acid molecule and the nucleic acid probe.

25. The method of claim 24, wherein the DNA:

RNA hybrid is bound by a DNA;

RNA hybrid-binding antibody.

26. The method of claim 25, wherein the DNA:

RNA hybrid-binding antibody is bound to a solid phase or adapted to be bound to a solid phase.

27. The method of claim 26, wherein the solid phase is a magnetic bead.

28. The method of claim 19, wherein the nucleic acid molecule is a viral nucleic acid molecule.

29. The method of claim 2, wherein the fixed biological sample is a cervical sample.

30. The method of claim 9, wherein the detergent is deoxycholate, 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100).

31. The method of claim 2, wherein the composition comprises Tris-HCl, diethanolamine, Brij-58, and sodium azide.

32. The method of claim 31, wherein the composition has a pH of approximately 9.4.

33. The method of claim 31, wherein the amount of each of Tris-HCl and diethanolamine is about 150 mM or greater.