Compositions and methods for purifying nucleic acids from stabilization reagents

Abstract

The invention features reagents, methods and kits for the purification of RNA, or DNA, or both, from a sample.

1 . A method for isolating substantially pure and undegraded RNA from a test sample containing RNA, comprising: 
 (a) contacting a sample with a Solubilization Solution comprising a buffer at a pH between about 7 and 9, a base, an amphiphillic reagent;    (b) contacting the sample with a Lysis Solution buffered at a pH of greater than about 7 to create an isolation sample, wherein the Lysis Solution comprises a complexing salt;    (c) contacting the isolation sample to a solid support such that nucleic acids comprising substantially undegraded RNA in the isolation sample bind to the solid support;    (d) washing the solid support with one or more Wash Solutions to remove materials other than bound nucleic acids comprising substantially undegraded RNA; and    (e) eluting the bound substantially undegraded RNA from the solid support in order to obtain substantially pure and undegraded RNA.    
     
     
         2 . A method for isolating DNA from a test sample containing DNA, comprising: 
 (a) contacting a sample with a Solubilization Solution comprising a buffer comprising a buffer at a pH between about 7 and 9, a base, an amphiphillic reagent;    (b) contacting the sample with a Lysis Solution buffered at a pH of greater than about 7 to create an isolation sample, wherein the Lysis Solution comprises a complexing salt;    (c) contacting the isolation sample with either (i) a Binding Solution comprising a buffer, a lithium salt, and an amphiphillic reagent, or (ii) a wash solution comprising a lithium salt and an alcohol to create a binding sample;    (d) contacting the binding sample to a solid support such that nucleic acids comprising substantially undegraded DNA in the binding sample bind to the solid support;    (e) washing the solid support with one or more Wash Solutions to remove materials other than bound nucleic acids comprising substantially undegraded DNA; and    (f) eluting the bound substantially undegraded DNA from the solid support in order to obtain substantially pure and undegraded DNA.    
     
     
         3 . The method of  claim 2 , wherein the test sample is a pellet.  
     
     
         4 . The method of  claim 2 , wherein the buffer in the Solubilization Solution is Tris HCl.  
     
     
         5 . The method of  claim 4 , wherein the buffer in the Solubilization Solution is present at a concentration of about 10 to 20 mM.  
     
     
         6 . The method of  claim 2 , wherein the base in the Solubilization Solution is Tris base present at a concentration of about 20 to 50 mM.  
     
     
         7 . The method of  claim 2 , wherein the Solubilization Solution further comprises an amphiphillic reagent.  
     
     
         8 . The method of  claim 7 , wherein the amphiphillic reagent is a detergent.  
     
     
         9 . The method of  claim 8 , wherein the detergent is a non-ioninc, anionic, cationic or zwitterionic detergent.  
     
     
         10 . The method of  claim 9 , wherein the non-ionic detergent is from the Tween class, Triton class, Tergitols, Nonidets or Igepal.  
     
     
         11 . The method of  claim 10 , wherein the detergent is present at a concentration of about 5 to 15%.  
     
     
         12 . The method of  claim 2 , wherein the Solubilization Solution further comprises a chelator.  
     
     
         13 . The method of  claim 12 , wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).  
     
     
         14 . The method of  claim 13 , wherein the chelating agent is present at a concentration of about 1 to 20 mM.  
     
     
         15 . A method for isolating substantially pure and undegraded RNA from a test sample containing RNA, comprising: 
 (a) contacting the test sample with guanidinium stabilization agent and with an alcohol;    (b) Centrifuging the test sample to form crude lysate pellet and a supernatant;    (c) removing the supernatant;    (d) contacting the crude lysate with a Lysis Solution buffered at a pH of greater than about 7 to create an isolation sample, wherein the Lysis Solution comprises a complexing salt;    (e) contacting the isolation sample to a solid support such that nucleic acids comprising substantially undegraded RNA in the isolation sample bind to the solid support;    (f) washing the solid support with one or more Wash Solutions to remove materials other than bound nucleic acids comprising substantially undegraded RNA; and    (g) eluting the bound substantially undegraded RNA from the solid support in order to obtain substantially pure and undegraded RNA.    
     
     
         16 . The method of  claim 2 , wherein the Lysis Solution further comprises a buffer, an amphiphillic reagent, and a complexing salt, but is free of strong chaotropic substances.  
     
     
         17 . The method of  claim 16 , wherein the chaotropic substance is guanidinium salt and/or urea.  
     
     
         18 . The method of  claim 16 , wherein the buffer is Tris-HCl.  
     
     
         19 . The method of  claim 16 , wherein the pH of the Lysis Solution is at least about 8.  
     
     
         20 . The method of  claim 16 , wherein the pKa of the buffer is at least about 8.  
     
     
         21 . The method of  claim 16 , wherein the buffer is present at a concentration of about 50 to 150 mM.  
     
     
         22 . The method of  claim 16 , further comprising a base.  
     
     
         23 . The method of  claim 22 , wherein the base is alkali-metal hydroxide.  
     
     
         24 . The method of  claim 23 , wherein the alkali-metal hydroxide is sodium hydroxide, potassium hydroxide, or lithium hydroxide.  
     
     
         25 . The method of  claim 16 , wherein the complexing salt is an alkali-metal salt.  
     
     
         26 . The method of  claim 25 , wherein the alkali-metal salt is a lithium salt.  
     
     
         27 . The method of  claim 26 , wherein the lithium salt is lithium chloride or lithium bromide.  
     
     
         28 . The method of  claim 26 , wherein the alkali-metal salt is present at a concentration between 3 and 10 M.  
     
     
         29 . The method of  claim 16 , wherein the amphiphillic reagent is a detergent.  
     
     
         30 . The method of  claim 29 , wherein the detergent is a non-ioninc, anionic, cationic or zwitterionic detergent.  
     
     
         31 . The method of  claim 30 , wherein the non-ionic detergent is from the Tween class, Triton class, Tergitols, Nonidets or Igepal.  
     
     
         32 . The method of  claim 29 , wherein the detergent is present at a concentration of about 5 to 15%.  
     
     
         33 . The method of  claim 16 , wherein the amphiphillic reagent is a surfactant.  
     
     
         34 . The method of  claim 33 , wherein the surfactant is diethylene glycol monoethyl ether (DGME).  
     
     
         35 . The method of  claim 33 , wherein the surfactant is present at a concentration of about 5 to 15%.  
     
     
         36 . The method of  claim 16 , wherein the amphiphillic reagent is a combination of one or more detergents and/or one or more surfactants.  
     
     
         37 . The method of  claim 16 , wherein the Lysis Solution further comprises a chelating agent.  
     
     
         38 . The method of  claim 37 , wherein the chelating agent is present at a concentration of about 1 to 100 mM.  
     
     
         39 . The method of  claim 37 , wherein the chelating agent is EDTA or CDTA.  
     
     
         40 . The method of  claim 2 , further comprising contacting the sample with a Proteinase K Solution comprising Proteinase K.  
     
     
         41 . The method of  claim 40 , wherein the Proteinase K is present at a concentration of about 10 to 25 mg/mL.  
     
     
         42 . The method of  claim 2 , wherein the Binding Solution comprises a buffer, a complexing salt, and an amphiphilic reagent.  
     
     
         43 . The method of  claim 42 , wherein the buffer in the Binding Solution is a Tris buffer at a pH of at least about 7.  
     
     
         44 . The method of  claim 42 , wherein the buffer in the Binding Solution is present at a concentration of about 50 to 150 mM.  
     
     
         45 . The method of  claim 42 , wherein the Binding Solution further comprises a base to adjust the pH of the Binding Solution to no less that 7.  
     
     
         46 . The method of  claim 43 , wherein the base is an alkali-metal hydroxide.  
     
     
         47 . The method of  claim 44 , wherein the alkali-metal hydroxide is sodium hydroxide, potassium hydroxide, or lithium hydroxide.  
     
     
         48 . The method of  claim 42 , wherein the complexing salt is an alkali metal salt.  
     
     
         49 . The method of  claim 48 , wherein the alkali metal salt is a sodium salt or lithium salt  
     
     
         50 . The method of  claim 49 , wherein the alkali metal salt is lithium chloride or lithium bromide.  
     
     
         51 . The method of  claim 42 , wherein the complexing salt is present at a concentration of between about 5 to 15 M.  
     
     
         52 . The method of  claim 42 , wherein the amphiphilic reagent is a detergent.  
     
     
         53 . The method of  claim 52 , wherein the detergent is a non-ioninc, anionic, cationic or zwitterionic detergent.  
     
     
         54 . The method of  claim 53 , wherein the non-ionic detergent is from the Tween class, Triton class, Tergitols, Nonidets or Igepal.  
     
     
         55 . The method of  claim 52 , wherein the detergent is present at a concentration of about 5 to 15%.  
     
     
         56 . The method of  claim 52 , wherein the amphiphillic reagent is a surfactant.  
     
     
         57 . The method of  claim 56 , wherein the surfactant is diethylene glycol monoethyl ether (DGME).  
     
     
         58 . The method of  claim 56 , wherein the surfactant is present at a concentration of about 5 to 15%.  
     
     
         59 . The method of  claim 42 , wherein the amphiphillic reagent in the Binding Solution is a combination of one or more detergents and/or one or more surfactants.  
     
     
         60 . The method of  claim 2 , wherein the alcohol in the one or more Wash Solutions is present at a concentration greater than 50%.  
     
     
         61 . The method of  claim 2 , wherein the alcohol in the one or more Wash Solutions is ethanol or methanol.  
     
     
         62 . The method of  claim 2 , wherein a first Wash Solution (Wash Solution I) contains an alkali metal salt at a concentration of about 4 to 10 M.  
     
     
         63 . The method of  claim 62 , wherein the alkali metal salt is a sodium or lithium salt.  
     
     
         64 . The method of  claim 63 , wherein the alkali metal salt is lithium chloride or lithium bromide.  
     
     
         65 . The method of  claim 62 , wherein the Wash Solution I further comprises an alcohol at a concentration of about 25 to 80%.  
     
     
         66 . The method of  claim 65 , wherein the alcohol is ethanol or methanol.  
     
     
         67 . The method of  claim 1 , wherein a second Wash Solution (Wash Solution II) comprises a buffer at a pH of about 6 to 8 and an alcohol at a concentration of about 50 to 90%.  
     
     
         68 . The method of  claim 67 , wherein the alcohol is ethanol or methanol.  
     
     
         69 . The method of  claim 67 , wherein the buffer is Tris-HCl at a concentration of bout 50 to 150 mM.  
     
     
         70 . The method of  claim 67 , further comprising a chelator.  
     
     
         71 . The method of  claim 70 , wherein the chelator is EDTA or CDTA at a concentration of about 1 to 20 mM.  
     
     
         72 . The method of  claim 1 , wherein one or more Wash Solutions is a DNase Wash Solution comprises an alcohol at a concentration of about 10 to 50%, an alkali metal salt at a concentration of about 2 to 5 M, and a chelating agent at a concentration of about 25 to 100 mM.  
     
     
         73 . The method of  claim 72 , wherein the alcohol is ethanol or methanol.  
     
     
         74 . The method of  claim 72 , wherein the chelating agent is EDTA, CDTA or citrate.  
     
     
         75 . The method of  claim 72 , wherein the alkali metal salt is a lithium salt  
     
     
         76 . The method of  claim 75 , wherein the lithium salt is lithium chloride or lithium bromide.  
     
     
         77 . The method of  claim 2 , wherein the solid support comprises components of silica, cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, or polyvinylidene fluoride, or combinations thereof.  
     
     
         78 . The method of  claim 77 , wherein the solid support is pre-treated with RNase solution prior to contacting the biological material with the solid support.  
     
     
         79 . The method of  claim 15 , wherein the alcohol in step (a) is either ethanol or methanol, or a combination of methanol and ethanol.  
     
     
         80 . The method of  claim 15 , wherein the alcohol in step (a) is present in a concentration of between about 30% and 100%.  
     
     
         81 . The method of  claim 15 , wherein the alcohol in step (a) is present in a concentration of between 70% or 95%.  
     
     
         82 . The method of  claim 15 , wherein the guanidinium stabilizing agent is Tempus™ stabilizing agent.  
     
     
         83 . A method for isolating both DNA and RNA from a sample comprising: 
 (a) dividing the sample into a first and second tube;    (b) isolating the RNA from the sample in the first tube according to the method of  claim 1;  and    (c) isolating the DNA from the sample in the second tube according to the method of  claim 2 .    
     
     
         84 . A method for isolating both DNA and RNA from a sample comprising: 
 (a) solubilizing the sample with a Solubilization Solution comprising a buffer at a pH between about 7 and 9, a base, an amphiphillic reagent;    (b) dividing the sample into a first and second tube;    (c) isolating the RNA from the sample in the first tube according to the method comprising 
 (i) contacting the sample with a Lysis Solution buffered at a pH of greater than about 7 to create an isolation sample, wherein the Lysis Solution comprises a complexing salt;  
 (ii) contacting the isolation sample to a solid support such that nucleic acids comprising substantially undegraded RNA in the isolation sample bind to the solid support;  
 (iii) washing the solid support with one or more Wash Solutions to remove materials other than bound nucleic acids comprising substantially undegraded RNA; and  
 (iv) eluting the bound substantially undegraded RNA from the solid support in order to obtain substantially pure and undegraded RNA; and  
   (d) isolating the DNA from the sample in the second tube according to the method comprising 
 (i) contacting the sample with a Lysis Solution buffered at a pH of greater than about 7 to create an isolation sample, wherein the Lysis Solution comprises a complexing salt;  
 (ii) contacting the isolation sample with either (1) a Binding Solution comprising a buffer, a lithium salt, and an amphiphillic reagent, or (2) a wash solution comprising a lithium salt and an alcohol to create a binding sample;  
 (iii) contacting the binding sample to a solid support such that nucleic acids comprising substantially undegraded DNA in the binding sample bind to the solid support;  
 (iv) washing the solid support with one or more Wash Solutions to remove materials other than bound nucleic acids comprising substantially undegraded DNA; and  
 (v) eluting the bound substantially undegraded DNA from the solid support in order to obtain substantially pure and undegraded DNA.  
   
     
     
         85 . A formulation for solubilizing a material containing nucleic acids, comprising 
 (a) Tris-HCl at a concentration of about 10 to 20 mM and at a pH between about 7 and 9,    (b) Tris base at a concentration of about 20 to 50 mM,    (c) Triton-X at a concentration of about 5 to 15%, and    (d) EDTA at a concentration of about 1 to 20 mM.    
     
     
         86 . A kit for isolating RNA, DNA, or both, which comprises packaging, containing, separately packaged: 
 (a) a Solubilization Solution;    (b) a Lysis Solution;    (c) a Wash I Solution or Binding Solution;    (d) a Wash II Solution; and    (e) a protocol for isolation of RNA, DNA, or both, from a sample.    
     
     
         87 . A method for isolating substantially pure and undegraded RNA from blood sample containing RNA, comprising: 
 (a) contacting the blood sample with Tempus™ stabilizing agent and with ethanol or methanol;    (b) centrifuging the test sample to form a crude lysate pellet and a supernatant;    (c) removing the supernatant;    (d) contacting the crude lysate pellet with a Lysis Solution buffered at a pH of greater than about 7 to create an isolation sample, wherein the Lysis Solution comprises a complexing salt;    (e) contacting the isolation sample to a solid support such that nucleic acids comprising substantially undegraded RNA in the isolation sample bind to the solid support;    (f) washing the solid support with one or more Wash Solutions to remove materials other than bound nucleic acids comprising substantially undegraded RNA; and    (g) eluting the bound substantially undegraded RNA from the solid support in order to obtain substantially pure and undegraded RNA.