Separation and Purification of Nucleic Acid from Paraffin-containing Samples

Abstract

Disclosed are rapid and reproducible methods for separating and purifying nucleic acids from paraffin-containing tissue samples. The disclosed methods involve a melting step, where paraffin-containing tissue samples are heated in the presence of a detergent, causing the paraffin to melt and tissue sample cells to lyse. From the resulting two-phase mixture (i.e., a paraffin phase and an aqueous phase), the aqueous phase is collected for purification of nucleic acid. Protease(s) can be used at one or more points in the separation process to facilitate tissue cell lysis and/or degrade proteins that could degrade nucleic acid or interfere with subsequent genetic manipulation or analysis.

1 - 45 . (canceled)  
     
     
         46 . A method for separating deoxyribonucleic acid from a paraffin-containing sample comprising: 
 (a) heating said sample at about 75-100° C. for about 1-60 minutes in the presence of a detergent to produce a paraffin phase and an aqueous phase;    (b) removing said aqueous phase from said paraffin phase; and    (c) adding a protease to said aqueous phase and incubating said aqueous phase at about 25-80° C. for about 5-60 minutes.    
     
     
         47 . The method of  claim 46 , wherein said paraffin-containing sample is a formalin-fixed paraffin embedded (FFPE) sample.  
     
     
         48 . The method of  claim 46 , wherein said heating is at about 85-100° C.  
     
     
         49 . The method of  claim 46 , wherein said heating is at about 100° C.  
     
     
         50 . The method of  claim 46 , wherein said heating is about 1-30 min.  
     
     
         51 . The method of  claim 46 , wherein said heating is for about 10 min.  
     
     
         52 . The method of  claim 46 , wherein said detergent is an ionic detergent.  
     
     
         53 . The method of  claim 52 , wherein said ionic detergent is sodium dodecyl sulfate (SDS) or Sarcosine.  
     
     
         54 . The method of  claim 46 , wherein said detergent is a non-ionic detergent.  
     
     
         55 . The method of  claim 54 , wherein said non-ionic detergent is Triton X-114, NP-40 or Tween-20.  
     
     
         56 . The method of  claim 46 , wherein said protease is Proteinase K.  
     
     
         57 . The method of  claim 46 , wherein said incubating is at about 35-70° C.  
     
     
         58 . The method of  claim 46 , wherein said incubating is at about 55-65° C.  
     
     
         59 . The method of  claim 46 , wherein said incubating is at about 62° C.  
     
     
         60 . The method of  claim 46 , wherein said incubating is for about 5-30 min.  
     
     
         61 . The method of  claim 46 , wherein said incubating is for about 10 min.  
     
     
         62 . The method of  claim 46 , further comprising purifying said deoxyribonucleic acid from said aqueous phase.  
     
     
         63 . The method of  claim 62 , wherein said purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch® purification, or nucleic acid hybridization.  
     
     
         64 . A method for separating deoxyribonucleic acid from a paraffin-containing sample, comprising: 
 (a) heating said sample to about 75-100° C. for about 1-60 minutes in the presence of a detergent to produce a paraffin phase and an aqueous phase; and    (b) adding a protease to said aqueous phase and incubating said aqueous phase at about 75-100° C. for about 5-60 minutes.    
     
     
         65 . The method of  claim 64 , wherein said paraffin-containing sample is a formalin-fixed paraffin embedded (FFPE) sample.  
     
     
         66 . The method of  claim 64 , wherein said heating is about 85-100° C.  
     
     
         67 . The method of  claim 64 , wherein said heating is about 100° C.  
     
     
         68 . The method of  claim 64 , wherein said heating is for about 1-30 min.  
     
     
         69 . The method of  claim 64 , wherein said heating is for about 10 min.  
     
     
         70 . The method of  claim 64 , wherein said detergent is an ionic detergent.  
     
     
         71 . The method of  claim 70 , wherein said ionic detergent is sodium dodecyl sulfate (SDS) or Sarcosine.  
     
     
         72 . The method of  claim 64 , wherein said detergent is a non-ionic detergent.  
     
     
         73 . The method of  claim 72 , wherein said non-ionic detergent is Triton X-114, NP-40 or Tween-20.  
     
     
         74 . The method of  claim 64 , wherein said protease is Proteinase K.  
     
     
         75 . The method of  claim 64 , wherein said incubating is at about 35-70° C.  
     
     
         76 . The method of  claim 64 , wherein said incubating is at about 55-65° C.  
     
     
         77 . The method of  claim 64 , wherein said incubating is at about 62° C.  
     
     
         78 . The method of  claim 64 , wherein said incubating is for about 5-30 min.  
     
     
         79 . The method of  claim 64 , wherein said incubating is for about 10 min.  
     
     
         80 . The method of  claim 64 , further comprising purifying said deoxyribonucleic acid from said aqueous phase.  
     
     
         81 . The method of  claim 80 , wherein said purifying involves TRIZOL precipitation, guanidinium isothiocyanate, anion exchange chromatography, silica-based purification, ChargeSwitch® purification, or nucleic acid hybridization.