Alkaline shock-based preparation of nucleic acids
View Patent ↗Method of preparing a biological sample appropriate for use in a subsequent in vitro nucleic acid amplification reaction. The method involves a rapid, transient exposure to alkaline conditions which can be achieved by mixing an alkaline solution with a pH-buffered solution that includes a detergent and the biological sample to be tested for the presence of particular nucleic acid species using in vitro amplification. The invented method advantageously can improve detection of some target nucleic acids without substantially compromising detectability of others. The method is particularly useful for simultaneously preparing RNA and DNA templates that can be used in multiplex amplification reactions.
1. A method of processing a biological sample, comprising the steps of:
(a) combining said biological sample with a pH buffer and a detergent that lyses or disrupts biological membranes that may be present in said biological sample, whereby there is created a first liquid composition having a first pH;
(b) mixing with said first liquid composition an alkaline composition, whereby there is created a second liquid composition having a second pH,
wherein said alkaline composition is an aqueous solution comprising a strong base at a concentration of at least 0.1 N,
wherein said second pH is at least 0.2 pH units higher than said first pH, and
wherein said second pH is lower than pH 9.5; and
(c) purifying nucleic acids from said second liquid composition.
2. The method of claim 1 , wherein purifying step (c) comprises,
(i) immobilizing nucleic acids from said second liquid composition onto a solid support by sequence-specific target capture; and
(ii) washing the solid support to remove any material not immobilized thereon.
3. The method of claim 1 , wherein purifying step (c) comprises,
(i) immobilizing nucleic acids from said second liquid composition onto a solid support by non-specific target capture that is substantially independent of base sequence; and
(ii) washing the solid support to remove any material not immobilized thereon.
4. The method of claim 1 , wherein purifying step (c) comprises immobilizing onto a solid support nucleic acids from said second liquid composition, and then washing the solid support to remove any material not immobilized thereon.
5. The method of claim 4 , wherein the step for immobilizing onto the solid support comprises immobilizing by sequence-specific target capture.
6. The method of claim 4 , wherein the step for immobilizing onto the solid support comprises immobilizing by non-specific target capture that is substantially independent of base sequence.
7. The method of claim 4 , wherein the nucleic acids purified in step (c) comprise RNA.
8. The method of claim 4 , wherein the nucleic acids purified in step (c) comprise RNA and DNA.
9. The method of claim 4 , wherein the nucleic acids purified in step (c) comprise DNA.
10. The method of claim 4 , further comprising the step of performing an in vitro nucleic acid amplification reaction using the nucleic acids purified in step (c) as templates.
11. The method of claim 10 , wherein said in vitro nucleic acid amplification reaction comprises a reverse transcriptase.
12. The method of claim 10 , wherein said in vitro nucleic acid amplification reaction is a multiplex reaction.
13. The method of claim 10 , wherein the in vitro nucleic acid amplification reaction is an isothermal in vitro nucleic acid amplification reaction.
14. The method of claim 4 , wherein said solid support comprises paramagnetic monodisperse particles.
15. The method of claim 14 , further comprising the step of performing an in vitro nucleic acid amplification reaction using the nucleic acids purified in step (c) as templates.
16. The method of claim 15 , wherein said in vitro nucleic acid amplification reaction comprises a reverse transcriptase.
17. The method of claim 15 , wherein said in vitro nucleic acid amplification reaction is a multiplex reaction.
18. The method of claim 15 , wherein the in vitro nucleic acid amplification reaction is an isothermal in vitro nucleic acid amplification reaction.
19. The method of claim 4 , wherein said second pH is in the range of from pH 8.0 to 9.2.
20. The method of claim 19 , wherein said strong base is selected from the group consisting of NaOH and LiOH.
21. The method of claim 1 , further comprising the step of performing an in vitro nucleic acid amplification reaction using the nucleic acids purified in step (c) as templates.
22. The method of claim 21 , wherein said in vitro nucleic acid amplification reaction comprises a reverse transcriptase.
23. The method of claim 21 , wherein said in vitro nucleic acid amplification reaction is a multiplex reaction.
24. The method of claim 21 , wherein the in vitro nucleic acid amplification reaction is an isothermal in vitro nucleic acid amplification reaction.
25. The method of claim 21 , wherein the nucleic acids purified in step (c) comprise RNA.
26. The method of claim 21 , wherein the nucleic acids purified in step (c) comprise RNA and DNA.
27. The method of claim 21 , wherein the nucleic acids purified in step (c) comprise DNA.