Pretreatment method for extraction of nucleic acid from biological samples and kits therefor

Abstract

The present invention relates to methods for pretreating biological samples for extraction of nucleic acid therefrom. The present invention employs a combination of at least one protein denaturant with one or more of the following elements to form a reaction mixture for extraction of nucleic acid: (1) at least one aprotic solvent, (2) stepwise heating, and (3) sample dilution.

1 . A method of treating a biological sample for extraction of nucleic acid therefrom comprising mixing the sample with at least one protein denaturant and heating the mixture stepwise. 
     
     
         2 . The method of  claim 1  wherein the stepwise heating is in a temperature range of about 55° C. to about 85° C. 
     
     
         3 . A method of treating a biological sample for extraction of nucleic acid therefrom comprising treating the sample with at least one protein denaturant to form a reaction mixture and diluting the reaction mixture with a diluent. 
     
     
         4 . The method of  claim 3  wherein the diluent is selected from the group consisting of water, aqueous buffer solutions and aprotic solvents. 
     
     
         5 . The method of  claim 3  wherein the method is carried out at a temperature at or above about 4° C. 
     
     
         6 . The method of  claim 5  wherein the method is carried out in a temperature range of about 25° C. to about 95° C. 
     
     
         7 . A reaction mixture for treating a biological sample for extraction of nucleic acid therefrom, wherein said reaction mixture comprises at least one protein denaturant and at least one aprotic solvent. 
     
     
         8 . The reaction mixture of  claim 7  wherein the protein denaturant is selected from the group consisting of proteolytic enzymes, detergents, surfactants, solvents, amides, reducing agents, bases, protein denaturing salts and combinations thereof. 
     
     
         9 . The reaction mixture of  claim 8  wherein the protein denaturant is a proteolytic enzyme selected from the group consisting of proteinase K, pronase, pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase. 
     
     
         10 . The reaction mixture of  claim 9  wherein the proteolytic enzyme is proteinase K. 
     
     
         11 . The reaction mixture of  claim 8  wherein the protein denaturant is a detergent selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, polyethylene glycol sorbitan monolaurate, polyethylene glycol sorbitan monooleate, NP-40, dodecyl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and polyethylene glycol tert-octylphenyl ether. 
     
     
         12 . The reaction mixture of  claim 8  wherein the protein denaturant is a surfactant. 
     
     
         13 . The reaction mixture of  claim 8  wherein the protein denaturant is a solvent selected from the group consisting of phenol, chloroform and isoamylalcohol. 
     
     
         14 . The reaction mixture of  claim 8  wherein the protein denaturant is an amide selected from the group consisting of N-ethylacetamide, N-butylacetamide and N,N-dimethylacetamide. 
     
     
         15 . The reaction mixture of  claim 8  wherein the protein denaturant is a reducing agent selected from the group consisting of glutathione, β-mercatoethanol and dithiothreitol. 
     
     
         16 . The reaction mixture of  claim 8  wherein the protein denaturant is a base selected from the group consisting of KOH, NaOH, NH 4 OH and Ca(OH) 2 . 
     
     
         17 . The reaction mixture of  claim 8  wherein the protein denaturant is a protein denaturing salt selected from the group consisting of NaCl, KCl, LiCl, NH 4 Cl, (NH 4 ) 2 SO 4  and perchlorate salt. 
     
     
         18 . The reaction mixture of  claim 7  wherein the aprotic solvent is selected from the group consisting of formamide, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetronitrile, benzene, toluene, acetone, cyclohexane, n-heptane, sulfur dioxide and hexamethylphosphoramide. 
     
     
         19 . The reaction mixture of  claim 18  wherein the aprotic solvent is formamide. 
     
     
         20 . The reaction mixture of  claim 7  wherein the treatment is carried out at a temperature at or above about 4° C. 
     
     
         21 . The reaction mixture of  claim 20  wherein the treatment is carried out in a temperature range of about 25° C. to about 95° C. 
     
     
         22 . The reaction mixture of  claim 20  wherein the treatment is carried out in a temperature range of about 70° C. to about 85° C. 
     
     
         23 . The reaction mixture of  claim 7  wherein the treatment is carried out by stepwise heating in a temperature range of about 55° C. to about 85° C. 
     
     
         24 . The reaction mixture of  claim 9  wherein the concentration of proteinase K is about 1 to about 100 units per milliliter of biological sample. 
     
     
         25 . The reaction mixture of  claim 20  wherein the concentration of formamide is about 10% to about 80% by volume. 
     
     
         26 . The reaction mixture of  claim 7  further comprising a solid support. 
     
     
         27 . The reaction mixture of  claim 26  wherein the solid support is selected from the group consisting of iron oxide, silica-coated particles, silica-coated membranes, glass fiber mats, glass membranes, glasses, zeolites and ceramics. 
     
     
         28 . A nucleic acid extracted from a biological sample, wherein said nucleic acid is extracted by treating the sample with at least one protein denaturant and at least one aprotic solvent. 
     
     
         29 . The nucleic acid of  claim 28  wherein the protein denaturant is selected from the group consisting of proteolytic enzymes, detergents, surfactants, solvents, amides, reducing agents, bases, protein denaturing salts and combinations thereof. 
     
     
         30 . The nucleic acid of  claim 29  wherein the protein denaturant is a proteolytic enzyme selected from the group consisting of proteinase K, pronase, pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase. 
     
     
         31 . The nucleic acid of  claim 30  wherein the proteolytic enzyme is proteinase K. 
     
     
         32 . The nucleic acid of  claim 29  wherein the aprotic solvent is selected from the group consisting of formamide, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetronitrile, benzene, toluene, acetone, cyclohexane and n-heptane, sulfur dioxide and hexamethylphosphoramide. 
     
     
         33 . The nucleic acid of  claim 32  wherein the aprotic solvent is formamide. 
     
     
         34 . The nucleic acid of  claim 28  wherein the nucleic acid is RNA.