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 composition which extracts nucleic acid from a tissue sample, the composition comprising a protease enzyme, a buffering component which maintains the pH of the composition at 7.5 or greater, and not more than 100 mM of a Ca 2+ -chelator wherein the composition does not contain a surface active agent and wherein the composition extracts nucleic acids suitable for PCR amplification following incubation with the tissue sample for not more than 60 minutes.  
   
   
       2 . The composition in accordance with  claim 1  wherein the protease enzyme is a subtilisin-like serine protease.  
   
   
       3 . The composition in accordance with  claim 2  wherein the proteinase K is present at a concentration of about 50 Units/ml or greater.  
   
   
       4 . The composition in accordance with  claim 3  wherein the proteinase K is present at a concentration of about 100 Units/ml or greater.  
   
   
       5 . The composition in accordance with  claim 1  wherein the extraction composition comprises not more than about 100 mM EDTA.  
   
   
       6 . The composition in accordance with  claim 5 , wherein the extraction composition comprises about 10 mM EDTA.  
   
   
       7 . The composition in accordance with  claim 1 , wherein the extraction composition contains a Tris-HCl buffer.  
   
   
       8 . The composition in accordance with  claim 7  wherein the Tris-HCl buffer is present at a concentration of about 80 mM or greater.  
   
   
       9 . The composition in accordance with  claim 1  wherein the extraction composition is buffered to a pH of at least 8.0 or greater.  
   
   
       10 . The composition in accordance with  claim 1  wherein the composition extracts nucleic acids suitable for PCR amplification incubation for not more than about 30 minutes.  
   
   
       11 . The composition in accordance with  claim 10  wherein the incubation is for not more than about 20 minutes.  
   
   
       12 . The composition in accordance with  claim 11  wherein the incubation is for not more than about 10 minutes.  
   
   
       13 . The composition in accordance with  claim 10  wherein the tissue sample comprises a plant tissue.  
   
   
       14 . The composition in accordance with  claim 10  wherein the plant tissue comprises a leaf or a seed or a portion thereof.  
   
   
       15 . The composition in accordance with  claim 14  wherein the plant tissue is ground.  
   
   
       16 . The composition in accordance with  claim 10  wherein the incubation is at a temperature of from about 37° C. to about 60° C.  
   
   
       17 . The composition in accordance with  claim 16  wherein the incubation is at a temperature of from about 50° C. to about 55° C.  
   
   
       18 . The composition in accordance with  claim 10  wherein the tissue sample comprises a mammalian tissue sample.  
   
   
       19 . The composition in accordance with  claim 18  wherein the mammalian tissue sample comprises a mouse tail tissue or a mouse ear tissue.  
   
   
       20 . The composition in accordance with  claim 18  wherein the incubation is at room temperature.