Nucleic acid purification method
The present invention relates to a method for purifying nucleic acids using a nucleic acid-binding phase which is furnished with a deficit of nucleic acid-binding groups A having a pK of 8 to 13, or which has groups A and binding-inhibiting groups N which are neutrally charged during the binding, and preferably during the elution, and the method comprises the following steps: (a) binding the nucleic acids to the nucleic acid-binding phase at a pH which is below the pH of the pK of the nucleic acid-binding groups A (binding pH); (b) eluting the nucleic acids at a pH which is above the binding pH (elution pH). In addition, corresponding kits and also nucleic acid-binding phases which can be used for purifying nucleic acids are disclosed. The technology according to the invention permits the purification of nucleic acids and, in particular, elution, with use of low salt concentrations, and so the purified nucleic acids can be directly processed, for example used in a PCR.
1. A method of purifying nucleic acids comprising:
(a) binding the nucleic acids to a nucleic acid-binding phase at a binding pH, the nucleic acid binding phase comprising nucleic acid binding groups having a pKa of from 8 to 13 and charge neutral groups having a neutral charge at the binding pH, wherein the binding pH is below the pKa of the nucleic acid-binding groups; and
(b) eluting the nucleic acids at an elution pH which is above the binding pH,
(i) wherein the nucleic acid biding groups are compounds selected from the group consisting of:
wherein
n is 1 to 5
R is a C 1 to C 6 alkyl group; and
* is amino, aminomethyl, aminoethyl, aminopropyl, dimethylamino, diethylamino, diisopropylamino, dipropylamino, diethanolamino, dipropanolamino, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, etheramine, polyetheramine, 4-diisobutylamino-1-butane, or 6-dipropylamino-1-hexane, and
(ii) wherein the charge neutral groups are compounds selected from the group consisting of
wherein
n is 1-5
R is a C1 to C6 alkyl group;
* is hydroxymethyl, hydroxyethyl, hydroxypropyl, ethanediol, propanediol, propanetriol, butanetriol, 3-glycidoxypropyl, ethyl glycidyl ether, or a C 1 to C 4 alkyl radical, or halogen.
2. The method according to claim 1 , wherein the nucleic acid-binding phase is attached to a support material, wherein only part of the support material comprises the nucleic acid binding groups.
3. The method according to claim 2 , wherein
the support material has a silica surface, the amount of nucleic acid binding groups being from 0.1 to 50 μmol.
4. The method according to claim 2 , wherein the nucleic acid-binding phase has a support material which complies with one or more of the following features:
(i) the support material is an oxidic material;
(ii) the support material is selected from the group consisting of Al 2 O 3 , TiO 2 , ZrO 2 , Ta 2 O 5 , SiO 2 and polysilicic acid;
(iii) the support material is SiO 2 or polysilicic acid;
(iv) the support material is an organic polymer selected from the group consisting of polystyrene and its derivatives, polyacrylate, polymethacrylate and its derivatives, polyurethane, nylon, polyethylene, polypropylene, polybutylidene and copolymers of these materials;
(v) the support material is a hydrogel;
(vi) the support material is glass or metal; and
(vii) the support material is magnetic.
5. The method according to claim 1 , wherein the binding in step (a) and/or the eluting in step (b) are carried out under conditions which comply with one or more of the following features:
(i) in the eluting in (b), the elution pH is below the pKa of the nucleic acid-binding groups;
(ii) the salt concentration in the binding and/or elution buffers is from 1 mM to 1000 mM;
(iii) the salt concentration is unchanged in the binding step (a) and/or in the eluting step (b) or is raised during the eluting;
(iv) the pH in the binding buffer is from pH 2 to pH 8,
(v) the pH in the elution buffer is from pH 2 to pH 10;
(vi) the temperature is unchanged in the binding step (a) and/or in the eluting step (b) or is raised during the eluting; and
(vii) the temperature in the eluting step is from 2° C. to 95° C.
6. The method according to claim 2 , wherein one or more of the following features is complied with:
(i) the support material was prepared with the nucleic acid binding groups and the charge neutral groups by silanization;
(ii) the nucleic acid binding groups and the charge neutral groups were introduced via reactive silanes;
(iii) the nucleic acid binding groups and the charge neutral groups were introduced by monofunctional, bi- or trifunctional reactive silanes or a mixture of at least two reactive silanes with different functionality;
(iv) the nucleic acid binding groups and the charge neutral groups were introduced by reactive silanes selected from the group consisting of aminosilanes, disilazanes, chlorosilanes and alkoxysilanes; and
(v) the proportion of nucleic acid binding groups based on the charge neutral groups, is from 1% to 99%.
7. The method according to claim 2 , wherein one or more of the following features is complied with:
(i) the support material was modified with starter molecules, with the nucleic acid binding groups and/or the charge neutral groups subsequently being introduced in the form of monomers;
(ii) the support material was modified with starter molecules, with said starter molecules having one or more of the following features:
(aa) the starter molecules are reactive silanes; and
(bb) the starter molecules are selected from the group consisting of
wherein X is halogen
Y1, Y2, Y3 or Y4 are, independently of one another, R, OR, OH or H and R is C1-C3 alkyl;
(iii) the starter molecule is 2-(chloromethyl)allyl trimethoxysilane;
(iv) the starter molecule is [3-(2-bromoisobutyryl)propyl]ethoxydimethylsilane;
(v) the nucleic acid binding groups were introduced by monomers selected from the group consisting of
N-(3-aminomethyl)methacrylamide, N-(3-aminoethyl)methacrylamide, N-(3-aminopropyl)methacrylamide, N-(3-aminoisopropyl)methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N,N-diisopropylacrylamide, N,N-(dimethylarnino)ethylacrylamide, N,N-(dimethylamino)ethyl acrylate, N,N-(dimethylamino)ethylmethacrylamide, N,N-(dimethylamino)ethyl methacrylate, N,N-(dimethylamino)propylacrylamide, N,N-(dimethylamino)-propyl acrylate, N,N-(dimethylamino)propylmethacrylamide, N,N-(dimethyl-amino)propyl methacrylate, N,N-(diethylamino)ethylacrylamide, N,N-(diethylamino)ethyl acrylate, N,N-(diethylamino)ethylmethacrylamide, N,N-(diethylamino)ethyl methacrylate, N,N-(diethylamino)propylacrylamide, N,N-(diethylamino)propyl acrylate, N,N-(diethylamino)propylmethacrylamide, N,N-(diethylamino)propyl methacrylate, N,N-(diisopropylamino)ethylacrylamide, N,N-(diisopropylamino)ethyl acrylate, N,N-(diisopropylamino)ethylmethacrylamide, N,N-(diisopropylamino)ethyl methacrylate, N,N-(diisopropylamino)-propylacrylamide, N,N-(diisopropylamino)propyl acrylate, N,N-(dimethylamino)propylmethacrylamide, N,N-(dimethylamino)propyl methacrylate, and 2-(diisopropylamino)ethyl methacrylate;
and
(vi) the charge neutral groups were introduced by monomers selected from the group consisting of butyl acrylate, propyl acrylate, ethyl acrylate, methyl acrylate, glycidyl methacrylate, hydroxyethyl methacrylate (HEMA), glycidoxypropyl methacrylate, glycerol mono-methacrylate (isomeric mixture), glycol mono-methacrylate, glycidyl acrylate, hydroxyethyl acrylate, glycidoxypropyl acrylate, glycerol mono-acrylate (isomeric mixture), glycol mono-acrylate and N-acryloxysuccinimide.
8. The method according to claim 2 , wherein the support material has starter groups which are at least partially functionalized with the nucleic acid binding groups being sterically shielded by the charge neutral groups.
9. The method according to claim 2 , wherein the amount of nucleic acid binding groups being from 0.1 to 10 μmol.
10. The method according to claim 5 , wherein the salt concentration in the binding and/or elution buffers is from 1 mM to 500 mM.
11. The method according to claim 5 , wherein the pH in the binding buffer is from pH 2 to pH 7.5.
12. The method according to claim 5 , wherein the pH in the elution buffer is from pH 4 to pH 10.
13. The method according to claim 5 , wherein the temperature in the eluting step is from 21° C. to 60° C.
14. The method according to claim 5 , wherein the salt concentration in the binding and/or elution buffers is from 1 mM to 250 mM.
15. The method according to claim 5 , wherein the pH in the binding buffer is from pH 4 to pH 8.
16. The method according to claim 5 , wherein the pH in the elution buffer is from pH 7 to pH 10.
17. The method according to claim 5 , wherein the salt concentration in the binding and/or elution buffers is from 1 mM to 100 mM.
18. The method according to claim 5 , wherein the pH in the binding buffer is from pH 4 to pH 7.5.
19. The method according to claim 5 , wherein the pH in the elution buffer is from pH 8 to pH 9.
20. The method according to claim 6 , wherein the proportion of nucleic acid binding groups based on the charge neutral groups, is from 1 to 50%.
21. The method according to claim 6 , wherein the proportion of nucleic acid binding groups based on the charge neutral groups, is from 1% to 25%.