Method for nucleic acids isolation
View Patent ↗Methods for isolating nucleic acids using a solid phase having alkylene sulfonyl-containing compounds on its surface are provided. In one embodiment, the method comprises: contacting a sample containing nucleic acids with a solid phase having alkylene sulfonyl-containing compounds on its surface in a first aqueous solution to provide a loaded solid phase; washing the loaded solid phase with a second aqueous solution to provide a washed solid phase; and eluting the washed solid phase with a low ionic strength liquid to obtain the isolated nucleic acids. Kits containing a solid phase having alkylene sulfonyl-containing compounds also are provided.
1. A method for isolating nucleic acids comprising:
contacting a sample containing nucleic acids with a solid phase in a first aqueous solution having a pH from about 6 to about 9 to provide a loaded solid phase; wherein the first aqueous solution comprises one or more salts; and the surface of the solid phase comprises one or more functional groups having structural formula (I):
wherein,
X is NR 1 , S, or O;
Y is —OH, —SH, —NHR 2 , —B(OH) 2 , or —B(OR 3 ) 2 ;
R 1 is hydrogen or alkyl;
R 2 is hydrogen, alkyl, or aryl;
each R 3 is independently alkyl or aryl, or alternatively, two of R 3 together with the atoms to which they are bonded from a heterocyclyl; and
represents the point of attachment to the solid phase;
washing the loaded solid phase with a second aqueous solution having a pH from about 6 to about 9 to provide a washed solid phase; wherein the second aqueous solution comprises a detergent and one or more salts; and
isolating the nucleic acids by eluting the washed solid phase with a low ionic strength liquid having a pH from about 5 to about 9 to obtain the isolated nucleic acids.
2. The method of claim 1 , wherein each alkylene is —CH 2 CH 2 —.
3. The method of claim 2 , wherein X is O, and Y is —OH.
4. The method of claim 1 , wherein the solid phase is in a form selected from the group consisting of particles, beads, microspheres, pellets, granules, spheroids, powder, sheets, chips, biochips, and combinations thereof.
5. The method of claim 1 , wherein the one or more salts are chaotropic salts, kosmotropic salts, or combinations thereof.
6. The method of claim 5 , wherein the chaotropic salts are salts containing an ion selected from the group consisting of SCN − , H 2 PO 4 − , HSO 4 − , HCO 3 − , I − , Cl − , NO 3 − , NH 4 + , Cs + , K − , guanidinium, alkyl ammonium, aryl ammonium, and a combination thereof.
7. The method of claim 5 , wherein the chaotropic salts are guanidinium chloride, guanidinium isocyanate, or a combination thereof.
8. The method of claim 1 , wherein eluting the washed solid phase comprises
contacting the washed solid phase with water or the low ionic strength solution; and
separating the washed solid phase from water or the low ionic strength solution to obtain the isolated nucleic acids.
9. The method of claim 5 , wherein the kosmotropic salts are salts containing an ion selected from the group consisting of SO 4 2− , HPO 4 2− , Mg 2+ , Ca 2+ , Li + , Na + , H + , OH − and HPO 4 2− .
10. The method of claim 5 , wherein the kosmotropic salts are selected from the group consisting of KCl, MgCl 2 , MgSO 4 , (NH 4 ) 2 SO 4 , NaH 2 PO 4 , Ca(Ac) 2 , (NH 4 ) 2 SO 4 , Cs 2 SO 4 , K 2 SO 4 , Na 2 SO 4 , Li 2 SO 4 , (NH 4 ) 2 HPO 4 , CsH 2 PO 4 , K 2 HPO 4 , NaH 2 PO 4 , LiH 2 PO 4 , NH 4 CO 2 CH 3 , CsCO 2 CH 3 , KCO 2 CH 3 , NaCO 2 CH 3 , NH 4 Cl, CsCl, KCl, NaCl, LiCl, NH 4 Br, CsBr, KBr, NaBr, and LiBr.
11. The method of claim 1 , wherein the first aqueous solution has a pH from about 7 to about 8.
12. The method of claim 1 , wherein the second aqueous solution has a pH from about 7 to about 8.
13. The method of claim 1 , wherein the low ionic strength liquid has a pH from about 6 to about 8.
14. The method of claim 1 , wherein the low ionic strength liquid is at a temperature from about room temperature to about 100° C.
15. The method of claim 1 , wherein the first and second aqueous solutions independently comprise one or more buffering agents.
16. The method of claim 1 , wherein the low ionic strength liquid comprises a buffering agent.
17. The method of claim 15 or 16 , wherein the one or more buffering agents are selected from the group consisting of 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)methylamine hydrochloride (TRIS HCl), N-tris(hydroxymethyl)methylglycine (Tricine), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinic acid (Cacodylate), 2-(N-morpholino)ethanesulfonic acid (MES), a phosphate salt, and a combination thereof.
18. The method of claim 15 or 16 , wherein the one or more buffering agents in the first and second aqueous solutions are independently present in a concentration from about 1 mM to about 2 M.
19. The method of claim 18 , wherein the one or more buffering agents in the first and second aqueous solutions are independently present in a concentration from about 10 mM to about 200 mM.
20. The method of claim 1 , wherein the first aqueous solution further comprises an alcohol or a polyalcohol.
21. The method of claim 1 , wherein the second aqueous solution further comprises an alcohol or a polyalcohol.
22. The method of claim 20 or 21 , wherein the alcohol is methanol, ethanol, or isopropanol.
23. The method of claim 20 or 21 , wherein the polyalcohol is glycerol, ethylene glycol, or a liquid polyethylene glycol.
24. The method of claim 1 , wherein the low ionic strength liquid further comprises a chelating agent.
25. The method of claim 1 , wherein the detergent is a cationic surfactant, a anionic surfactant, a non-ionic surfactant, an amphoteric surfactant, or a combination thereof.
26. The method of claim 1 , wherein the detergent is selected from the group consisting of lauryl sulfate, sodium dodecyl sulfate, polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100), and polysorbate 20.
27. The method of claim 1 , wherein the one or more salts in the first and second aqueous solutions are independently present in a concentration from about 0.1 M to about 6 M.
28. The method of claim 1 , wherein the one or more salts in the first and second aqueous solutions are independently present in a concentration from about 0.3 M to about 2 M.
29. The method of claim 1 , wherein the contact step comprises
mixing the sample containing nucleic acids with the first aqueous solution to form it mixture, and
adding the solid phase to the mixture.
30. The method of claim 1 , wherein the contact step comprises
mixing the solid phase with the first aqueous solution to form a mixture; and
adding the sample containing nucleic acids to the mixture.
31. The method of claim 1 , wherein the contact step comprises
mixing the sample containing nucleic acids with the solid phase to form a mixture; and
adding the first aqueous solution to the mixture.
32. The method of claim 1 , wherein the contacting step comprises
mixing the sample containing nucleic acids with the solid phase to form a mixture; and
adjusting the condition of the mixture to obtain the first aqueous solution containing the solid phase and the sample containing nucleic acids.
33. The method of claim 1 , wherein the washing step comprises
contacting the loaded solid phase with the second aqueous solution; and
separating the loaded solid phase from the second aqueous solution to obtain the washed solid phase.