Pretreatment method for extraction of nucleic acid from biological samples and kits therefor
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 to solubilize and extract nucleic acids therefrom comprising treating the sample with at least one protein denaturant and at least one aprotic solvent,
wherein the at least one 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,
wherein the at least one protein denaturant is a proteolytic enzyme selected from the group consisting of proteinase K, pronase, pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase, and
wherein the method is carried out at a temperature at or above 4° C.
2. The method of claim 1 wherein the at least one protein denaturant further comprises a second protein denaturant selected from the group consisting of proteolytic enzymes, detergents, surfactants, solvents, amides, reducing agents, bases, protein denaturing salts and combinations thereof.
3. The method of claim 1 wherein the proteolytic enzyme is proteinase K.
4. The method of claim 2 wherein the second 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 animonium bromide, 3[(3-cholamidipropyl)dimethylammonio]-1-propanesulfonate and polyethylene glycol tert-octylphenyl ether.
5. The method of claim 2 wherein the second protein denaturant is a surfactant.
6. The method of claim 2 wherein the second protein denaturant is a solvent selected from the group consisting of phenol, chloroform and isoamylalcohol.
7. The method of claim 2 wherein the second protein denaturant is an aniide selected from the group consisting of N-ethylacetamide, N-butylacetamide and N,N-dimethylacetamide.
8. The method of claim 2 wherein the second protein denaturant is a reducing agent selected from the group consisting of glutathione, β-mercatoethanol and dithiothreitol.
9. The method of claim 2 wherein the second protein denaturant is a base selected from the group consisting of KOH, NaOH, NH 4 OH and Ca(OH) 2 .
10. The method of claim 2 wherein the second 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 perchiorate salt.
11. The method of claim 1 wherein the at least one aprotic solvent is selected from the group consisting of dimethylacetamide, acetronitrile, beuzene, toluene, acetone, cyclohexane, n-heptane, sulfur dioxide and hexamethylphosphoramide.
12. The method of claim 1 wherein the aprotic solvent is fonnamide.
13. The method of claim 1 wherein the method is carried out in a temperature range of about 25° C. to about 95° C.
14. The method of claim 1 wherein the method is carried out in a temperature range of about 65° C. to about 85° C.
15. The method of claim 1 wherein the method is carried out by stepwise heating in a temperature range of about 55° C. to about 85° C.
16. The method of claim 3 wherein the concentration of proteinase K is about 1 to about 100 units per milliliter of biological sample.
17. The method of claim 1 wherein the biological sample is diluted or concentrated.
18. The method of claim 12 wherein the concentration of formamide is about 10% to about 80% by volume.
19. The method of claim 1 wherein the nucleic acid is RNA.
20. The method of claim 1 wherein a reagent is both a protein denaturant and an aprotic solvent selected from the group consisting of formamide, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetronitrile, benzene, toluene, acetone, cyclohexane, n-heptane, sulfur dioxide and hexamethylphosphoramide.
21. The method of claim 1 wherein the method comprises diluting the treated sample with a diluent selected from the group consisting of water, aqueous buffer solutions and aprotic solvents.
22. The method of claim 21 wherein the method is carried out in a temperature range of about 25° C. to about 95° C.
23. The method of claim 1 wherein the method further comprises the presence of a solid support.
24. The method of claim 23 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.
25. The method of claim 1 wherein the method further comprises diluting the treated sample with a diluent.