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 biological sample obtained from a plant, the method comprises incubating the sample in an extraction composition which contains a protease enzyme and a buffering component which buffers the composition to a pH of 7.5 or greater for an incubation time of not more than 30 minutes.  
   
   
       2 . The method in accordance with  claim 1  wherein the sample is incubated at room temperature.  
   
   
       3 . The method in accordance with  claim 1  wherein the sample is incubated at a temperature of at least about 37° C. and not more than about 60° C.  
   
   
       4 . The method in accordance with  claim 1  wherein the extraction composition does not contain a surface active agent.  
   
   
       5 . The method in accordance with  claim 1  wherein the protease enzyme is a subtilisin-like serine protease.  
   
   
       6 . The method in accordance with  claim 5  wherein the subtilisin-like protease is proteinase K.  
   
   
       7 . The method in accordance with  claim 6  wherein the proteinase K is present at a concentration of about 50 Units/ml or greater.  
   
   
       8 . The method in accordance with  claim 7  wherein the proteinase K is present at a concentration of about 100 Units/ml or greater.  
   
   
       9 . The method in accordance with  claim 1  wherein the extraction composition comprises not more than about 100 mM EDTA.  
   
   
       10 . The method in accordance with  claim 9 , wherein the extraction composition comprises about 10 mM EDTA.  
   
   
       11 . The method in accordance with  claim 1 , wherein the buffer component is Tris-HCl.  
   
   
       12 . The method in accordance with  claim 11 , wherein the Tris-HCl is present at a concentration of about 80 mM or greater.  
   
   
       13 . The method in accordance with  claim 1  wherein the extraction composition is buffered to a pH of at least 8.0 or greater.  
   
   
       14 . The method in accordance with  claim 1  wherein the incubating is for not more than about 20 minutes.  
   
   
       15 . The method in accordance with  claim 14  wherein the incubating is for not more than about 10 minutes.  
   
   
       16 . The method in accordance with  claim 1  wherein the biological sample obtained from a plant is plant tissue comprising a leaf or a seed or a portion thereof.  
   
   
       17 . The method in accordance with  claim 16  wherein the plant tissue is ground.