Alkylene glycols and polymers and copolymers thereof for direct isolation of nucleic acid from embedded samples

Abstract

Methods of directly isolating nucleic acid from an embedded biological sample are provided. An emulsified digest is generated in the presence of a thermostable protease, and an additive selected from an alkylene glycol, a poly(alkylene glycol), or a block copolymer having an average M n of 76 to 2900, or a salt or derivative or combination thereof. Nucleic acid is isolated directly from the emulsified digest. The methods eliminate the use of organic solvents such as xylene in a deparaffinization step prior to isolating nucleic acids from paraffin-embedded samples, for example.

1 . A method for isolation of nucleic acid from an embedded biological sample comprising:
 contacting the embedded biological sample with
 a) a thermostable protease, and 
 b) an additive comprising
 i) an alkylene glycol having an average M n  of 76 to 2900, 
 ii) a poly(alkylene glycol) having an average M n  of 76 to 2900, 
 iii) a copolymer having an average M n  of 76 to 2900, 
 iv) a salt of i), ii) or iii), 
 v) a derivative of i), ii) or iii), or 
 vi) any combination of i), ii) iii), iv) or v) 
 under conditions to provide an emulsified digest; and 
 
   isolating nucleic acid from the emulsified digest.   
     
     
         2 . The method of  claim 1  wherein the additive comprises an alkylene glycol, a combination of different alkylene glycols, or a salt or derivative or combination thereof. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1  wherein the additive comprises a poly(alkylene glycol), a combination of different poly(alkylene glycols), or a salt or derivative or combination thereof. 
     
     
         5 . The method of  claim 4  wherein the poly(alkylene glycol) comprises poly(propylene glycol), or a salt or derivative or combination thereof. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1  wherein the additive comprises a copolymer, or a salt or derivative or combination thereof. 
     
     
         8 . The method of  claim 7  wherein the copolymer is a block copolymer, or a salt or derivative, or combination thereof. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1  wherein the embedded biological sample is a paraffin-embedded sample. 
     
     
         11 . The method of  claim 1  wherein the embedded biological sample is a FFPE tissue sample. 
     
     
         12 . The method of  claim 1  wherein the conditions to provide an emulsified digest include heating to provide the emulsified digest. 
     
     
         13 . The method of  claim 1  wherein the conditions to provide an emulsified digest include use of a mild chaotrope during the contacting step. 
     
     
         14 . The method of  claim 1  wherein the conditions to provide an emulsified digest include heating in the presence of a mild chaotrope to provide the emulsified digest. 
     
     
         15 . The method of  claim 1 , wherein isolating nucleic acid comprises:
 contacting the emulsified digest with a solid support under conditions wherein nucleic acid binds to the solid support; and   eluting the nucleic acid from the solid support to provide eluted nucleic acid.   
     
     
         16 . The method of  claim 15 , wherein the nucleic acid is DNA and isolating DNA comprises:
 contacting the eluted nucleic acid with a ribonuclease to form a ribonuclease digest;   contacting the ribonuclease digest with a solid support under conditions wherein DNA binds to the solid support; and   eluting the DNA from the solid support.   
     
     
         17 . The method of  claim 15 , wherein the nucleic acid is RNA and isolating RNA comprises:
 contacting the eluted nucleic acid with a deoxyribonuclease to form a deoxyribonuclease digest;   contacting the deoxyribonuclease digest with a solid support under conditions wherein RNA binds to the solid support; and   eluting the RNA from the solid support.   
     
     
         18 . A kit, comprising:
 a) a thermostable protease, and   b) an additive comprising
 i) an alkylene glycol having an average M n  of 76 to 2900, 
 ii) a poly(alkylene glycol) having an average M n  of 76 to 2900, 
 iii) a copolymer having an average M n  of 76 to 2900, 
 iv) a salt of i), ii) or iii), 
 v) a derivative of i), ii) or iii), or 
 vi) any combination of i), ii) iii), iv) or v). 
   
     
     
         19 . The kit of  claim 18 , further comprising a digestion buffer. 
     
     
         20 . The kit of  claim 19 , wherein the digestion buffer comprises a mild chaotrope. 
     
     
         21 . The kit of  claim 18  wherein the additive comprises an alkylene glycol, a combination of different alkylene glycols, or a salt or derivative or combination thereof. 
     
     
         22 . The kit of  claim 21  wherein the alkylene glycol comprises propylene glycol, or a salt or derivative or combination thereof. 
     
     
         23 . The kit of  claim 18  wherein the additive comprises a poly(alkylene glycol), a combination of different poly(alkylene glycols) or a salt or derivative or combination thereof. 
     
     
         24 - 30 . (canceled)