Method and kit for sequential isolation of nucleotide species from a sample
View Patent ↗The invention provides a process and kit for serial isolation of DNA and RNA from the same sample. First, a siliceous solid support with preferential affinity for DNA over RNA is used to capture DNA in a lysate of a sample. Next, a siliceous solid support with similar affinity for RNA and DNA is used to capture RNA from the same lysate. The respective solid supports are recovered independent of each other, washed, and their bound nucleotide species are eluted. The invention further provides DNA and RNA prepared using the process in a minimal number of steps employing a minimal number of reagents. As the invention yields DNA and RNA of high quality and is amenable to automation, the invention may be used widely in the healthcare and pharmaceutical industries.
1. A method of serial recovery of two different nucleic acid species from a biological sample, comprising:
in a first binding step, homogenizing and enzymatically digesting the biological sample in a combined lysis and nucleic acid binding buffer, thereby generating a lysate comprising a first nucleic acid species and a second nucleic acid species, followed by selectively binding the first nucleic acid species to a first solid phase by contacting the lysate in the combined lysis and nucleic acid binding buffer with the first solid phase that selectively binds the first nucleic acid species;
separating the first solid phase with the bound first nucleic acid species from an unbound portion of the lysate;
in a second binding step, binding the second nucleic acid species to a second solid phase, different from the first solid phase, by contacting an unbound portion of the lysate in the combined lysis and nucleic acid binding buffer with the second solid phase that binds the first and second nucleic acid species;
enzymatically digesting the first nucleic acid species bound to the second solid phase; and
isolating the second nucleic acid species from the second solid phase.
2. The method according to claim 1 , wherein the combined lysis and nucleic acid binding buffer comprises a chaotropic salt solution or mildly acidic buffer.
3. The method according to claim 2 , wherein the chaotropic salt solution is guanidine HCl.
4. The method according to claim 2 , wherein the combined lysis and nucleic acid binding buffer comprises Proteinase K.
5. The method according to claim 1 , wherein the combined lysis and nucleic acid binding buffer is devoid of alcohols.
6. The method according to claim 1 , wherein the first nucleic acid species is deoxyribonucleic acid, DNA, and the second nucleic acid species is ribonucleic acid, RNA.
7. The method according to claim 1 , wherein the first solid support preferentially binds DNA over RNA when contacted with a mixture containing DNA and RNA.
8. The method according to claim 1 , wherein the second solid support binds RNA better than, or equally well as, DNA when contacted with a mixture containing DNA and RNA.
9. The method according to claim 1 , wherein the first and/or second solid support is composed of magnetic or superparamagnetic particles.
10. The method according to claim 1 , wherein the first and/or second solid support is composed of SiOH-coated magnetite crystals with >90% magnetite content in the size range 100-200 nm.
11. The method according to claim 1 , wherein the separated first nucleic acid species is:
washed in a slightly acidic buffer; and
eluted from the first solid support in a basic elution buffer.
12. The method according to claim 1 , wherein the separated second nucleic acid is:
washed in a slightly acidic buffer; and
eluted from the second solid support in a basic elution buffer.
13. The method according to claim 11 , wherein the basic elution buffer is devoid of alcohols.
14. The method according to claim 12 , wherein the washing buffer in the washing step for the second nucleic acid comprises DNase I.
15. The method according to claim 11 , wherein the washing buffer is devoid of alcohols.
16. A kit for use in a method for serial recovery of two different nucleic acid species from a biological sample, the kit comprising a combined lysis and nucleic acid binding buffer, a first solid phase that selectively binds a first nucleic acid species in the combined lysis and nucleic acid binding buffer, and a second solid phase that is different from the first solid phase and binds a second nucleic acid species and the first nucleic acid species in the combined lysis and nucleic acid binding buffer.
17. The kit according to claim 16 , wherein the combined lysis and nucleic acid binding buffer is devoid of alcohols.
18. The kit according to claim 16 , wherein the combined lysis and nucleic acid binding buffer is an aqueous solution of 7 M guanidine HCl, 50 mM Tris pH 7, 2% Tween 20.
19. The kit according to claim 16 , further comprising a wash buffer, a wash buffer for digestion of the first nucleic acid species, and an elution buffer.
20. The kit according to claim 19 , wherein the wash buffer, the wash buffer for digestion of the first nucleic acid species, and the elution buffer are devoid of alcohols.
21. The kit according to claim 19 , wherein the wash buffer is an aqueous solution of 10 mM Tris-HCl pH 6.5.
22. The kit according to claim 19 , wherein the wash buffer for digestion of the first nucleic acid species is an aqueous solution of 10 mM Tris-HCl pH 6-7 and 1-2 U DNase I.
23. The kit according to claim 19 , wherein the elution buffer is an aqueous solution of 10 mM Tris-HCl pH 8-10, and 1 mM EDTA.
24. The kit according to claim 16 , wherein the first and/or second solid support is composed of SiOH-coated magnetite crystals with >90% magnetite content in the size range 100-200 nm.
25. The kit according to claim 16 , where the first solid support preferentially binds DNA over RNA when contacted with a mixture of DNA and RNA.
26. The kit according to claim 16 , where the second solid support binds RNA better than, or equally well as, DNA when contacted with a mixture containing DNA and RNA.
27. The method according to claim 2 , wherein the combined lysis and nucleic acid binding buffer is devoid of alcohols.
28. The method according to claim 12 , wherein the basic elution buffer is devoid of alcohols.
29. The method according to claim 12 , wherein the washing buffer is devoid of alcohols.
30. The kit according to claim 22 , wherein the wash buffer for digestion of the first nucleic acid species is an aqueous solution of 10 mM Tris-HCl pH 6.5 and 1 U DNase I.
31. The kit according to claim 23 , wherein the elution buffer is an aqueous solution of 10 mM Tris-HCl pH 8.6 and 1 mM EDTA.