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

Abstract

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

What is claimed is: 
     
         1 . A method of lysing a fixed biological sample, the method comprising treating the fixed biological sample with a composition comprising a detergent, an amine, and a buffering agent. 
     
     
         2 . The method of  claim 1 , wherein the fixed biological sample is fixed with a cross-linking fixative agent. 
     
     
         3 . The method of  claim 2 , wherein the cross-linking fixative agent comprises an aldehyde. 
     
     
         4 . The method of  claim 3 , wherein the aldehyde is selected from the group consisting of formaldehyde and glutaraldehyde. 
     
     
         5 . The method of  claim 1 , wherein the fixed biological sample is fixed with a fixative agent comprising an alcohol. 
     
     
         6 . The method of  claim 5 , wherein the alcohol is selected from the group consisting of methanol and ethanol. 
     
     
         7 . The method of  claim 1 , wherein the fixative agent is selected from the group consisting of SUREPATH and PRESERVECYT. 
     
     
         8 . The method of  claim 1 , wherein the fixed biological sample is a cervical sample. 
     
     
         9 . 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.   
     
     
         10 . The method of  claim 9 , wherein the component is a nucleic acid molecule. 
     
     
         11 . The method of  claim 10  wherein the nucleic acid molecule comprises a specific sequence. 
     
     
         12 . The method of  claim 11 , 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. 
     
     
         13 . The method of  claim 12 , wherein the nucleic acid probe is adapted to be bound to a solid phase or is bound to a solid phase. 
     
     
         14 . The method of  claim 13 , wherein the solid phase is a magnetic bead. 
     
     
         15 . The method of  claim 12 , 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. 
     
     
         16 . The method of  claim 15 , wherein the DNA:RNA hybrid is bound by a DNA:RNA hybrid-binding antibody. 
     
     
         17 . The method of  claim 16 , wherein the DNA:RNA hybrid-binding antibody is bound to a solid phase or adapted to be bound to a solid phase. 
     
     
         18 . The method of  claim 17 , wherein the solid phase is a magnetic bead. 
     
     
         19 . The method of  claim 10 , wherein the nucleic acid molecule is a viral nucleic acid molecule. 
     
     
         20 . The method of  claim 9 , wherein the fixed biological sample is a cervical sample. 
     
     
         21 . The method according to  claim 9 , wherein the detergent is selected from the group consisting of polyoxyethyleneglycol dodecyl ether, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, Nonidet P-40, Igepal CA-630, deoxycholate, Triton X-100, sodium dodecyl sulfate, and polysorbate surfactants. 
     
     
         22 . The method according to  claim 9 , wherein the detergent is Triton-X100. 
     
     
         23 . The method according to  claim 9 , wherein the amine 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. 
     
     
         24 . The method according to  claim 9 , wherein the amine is diethanolamine. 
     
     
         25 . The method according to  claim 9 , 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-aminoethanesulfonic acid (“TES”); N-[tris(hydroxymethyl)methyl]-glycine (“TRICINE”); bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane (“bis-TRIS”); 1,3-bis[tris(hydroxymethyl)methylamino]propane (“bis-TRIS PROPANE”); carbonate-bicarbonate; glycine; phosphate; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (“HEPES”); N,N-bis(2-hydroxyethyl)glycine (“Bicine”); and 3-(N-morpholino)propanesulfonic acid (“MOPS”). 
     
     
         26 . The method according to  claim 9 , wherein the buffering agent is TRIS. 
     
     
         27 . The method according to  claim 9 , wherein the composition further comprises a preservative. 
     
     
         28 . The method according to  claim 19 , wherein the preservative is selected from the group consisting of sodium azide, gentomycin, 2-Methyl-4-isothiazolin-3-one, 5-Chloro-2-methyl-4-isothiazolin-3-one, and 1,2-Benzisothiazolin-3-one. 
     
     
         29 . The method according to  claim 19 , wherein the preservative is sodium azide. 
     
     
         30 . The method according to  claim 9  wherein the pH of the composition is 7 or greater. 
     
     
         31 . The method according to  claim 9 , wherein the composition further comprises at least one DNA-binding magnetic bead. 
     
     
         32 . The method according to  claim 9 , wherein the composition comprises:
 150 mM Tris-HCl;   300 mM diethanolamine;   3% Brij-58;   0.09% sodium azide;   
       wherein the composition has a pH of approximately pH 9.4.