Automatable method for nucleic acid isolation
The present invention relates to a highly automatable method for isolation and/or purification of nucleic acids from a biological sample, which is particularly suitable for nucleic acids-shorter than 250 bp and can be performed without a proteolytic pre-digestion step in an automated system, preferably a cartridge-based system. In a further aspect, the present invention also provides automated nucleic acid detection methods based on said isolation and/or purification method, as well as buffers and kits to be used in performing said methods.
1. A method for isolating and/or purifying nucleic acids from a nucleic acid-containing starting material, the method comprising: step (a) binding the nucleic acids to a nucleic acid binding support material by contacting the nucleic acid-containing starting material with the nucleic acid binding support in the presence of at least one chaotropic compound and at least one alcohol, wherein:
(i) the alcohol is an alcohol having 4 or 5 carbon atoms and is present at a concentration between about 17% (v/v) and about 50% (v/v), and in that
(ii) the at least one chaotropic compound is present at a concentration between about 1.5 M and about 4.5 M.
2. The method of claim 1 further comprising: step (b) eluting the bound nucleic acids from the nucleic acid binding support material with an elution buffer.
3. The method according to claim 1 , wherein no protease is added to step (a) or no incubation with a protease is performed directly before subjecting the nucleic acid-containing starting material to step (a).
4. The method according to claim 1 , wherein the alcohol having 4 or 5 carbon atoms is a monohydroxy alcohol.
5. The method according to claim 4 , wherein the monohydroxy alcohol is selected from the group consisting of n-butanol, sec-butanol, isobutanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl 1-butanol, 3-methyl 1-butanol, 2,2-dimethyl 1-propanol, 2- methyl 2-butanol, 3-methyl 2-butanol, or a mixture thereof.
6. The method according to claim 4 , wherein the monohydroxy alcohol is selected from the group consisting of n-butanol, 1-pentanol, sec-butanol, isobutanol, tert-butanol, or a mixture thereof.
7. The method according to claim 1 , wherein the alcohol having 4 or 5 carbon atoms is present in step (a) at a concentration between about 20% (v/v) and about 50% (v/v).
8. The method according to claim 1 , wherein the alcohol having 4 or 5 carbon atoms is present in step (a) at a concentration between about 35% (v/v) and about 40% (v/v).
9. The method according to claim 1 , wherein the at least one chaotropic compound is selected from thiocyanates, isocyanates, perchlorates, hydrochlorides, or mixtures thereof.
10. The method according to claim 1 , wherein the at least one chaotropic compound is guanidinium thiocyanate.
11. The method according to claim 1 , wherein the at least one chaotropic compound is present in step (a) at a concentration between about 2 M and about 4.5 M.
12. The method according to claim 1 , wherein the at least one chaotropic compound is present in step (a) at a concentration between about 3 M and about 3.5 M.
13. The method according to claim 1 , wherein the nucleic acid-containing starting material is a liquid biological sample selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid (CSF), and urine.
14. The method according to claim 1 , wherein said method is performed on an automated system on a removable cartridge.
15. An automated diagnostic method for detecting target nucleic acids, the method comprising the steps of:
(a) providing a nucleic acid-containing starting material into an automated system;
(b) isolating and/or purifying nucleic acids in said automated system, according to the method of claim 1 ;
(c) performing amplification in said automated system of target nucleic acids isolated and/or purified in step (b); and
(d) detecting the target nucleic acids amplified in step (c).
16. The automated diagnostic method according to claim 15 , wherein at least the steps (b) and (c) are performed on a cartridge.