Compositions and Methods for Nucleic Acid Extraction from Biological Samples

Abstract

Methods and compositions for extracting nucleic acids from a biological sample are provided. The extraction compositions contain a protease enzyme such as proteinase K at alkaline pH with little or no surfactant present. Extraction can be efficiently performed in 60 minutes or less at room temperature for certain mammalian tissue samples and at elevated temperatures for certain plant tissues.

1 . A method for extracting nucleic acids from a tissue sample, the method comprising incubating the sample in an extraction composition which is buffered to a pH of 7.5 or greater, wherein the extraction composition comprises a protease enzyme and does not contain a surface active agent and wherein the incubating is for sufficient time to release the nucleic acid but not more than about 60 minutes.  
     
     
         2 . The method in accordance with  claim 1  wherein the protease enzyme is a subtilisin-like serine protease.  
     
     
         3 . The method in accordance with  claim 2  wherein the subtilisin-like serine protease is proteinase K.  
     
     
         4 . The method in accordance with  claim 3  wherein the proteinase K is present at a concentration of about 50 Units/ml or greater.  
     
     
         5 . The method in accordance with  claim 4  wherein the proteinase K is present at a concentration of about 100 Units/ml or greater.  
     
     
         6 . The method in accordance with  claim 1  wherein the extraction composition comprises not more than about 100 mM EDTA.  
     
     
         7 . The method in accordance with  claim 6 , wherein the extraction composition comprises about 10 mM EDTA.  
     
     
         8 . The method in accordance with  claim 1 , wherein the extraction composition contains a Tris-HCl buffer.  
     
     
         9 . The method in accordance with  claim 8 , wherein the Tris-HCl buffer is present at a concentration of about 80 mM or greater.  
     
     
         10 . The method in accordance with  claim 1  wherein the extraction composition is buffered to a pH of at least 8.0 or greater.  
     
     
         11 . The method in accordance with  claim 1  wherein the incubating is for not more than about 30 minutes.  
     
     
         12 . The method in accordance with  claim 11  wherein the incubating is for not more than about 10 minutes.  
     
     
         13 . The method in accordance with  claim 1  wherein the biological sample comprises a plant tissue.  
     
     
         14 . The method in accordance with  claim 13  wherein the plant tissue comprises a leaf or a seed or a portion thereof.  
     
     
         15 . The method in accordance with  claim 14  wherein the plant tissue is ground.  
     
     
         16 . The method in accordance with  claim 13  wherein the incubating is at a temperature of from about 45° C. to about 60° C.  
     
     
         17 . The method in accordance with  claim 16  wherein the incubating is at a temperature of from about 50° C. to about 55° C.