Method for processing polynucleotide-containing samples
Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that retain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under a second set of conditions (e.g., higher temperature and/or more basic pH). The processing region can be used to, for example, concentrate polynucleotides of a sample and/or separate inhibitors of amplification reactions from the polynucleotides. Microfluidic devices with a processing region are disclosed.
1. A method for processing a polynucleotide-containing sample, the method comprising:
retaining polynucleotides from a sample on a plurality of binding particles in a process chamber under a first set of conditions, wherein the retaining step comprises binding the polynucleotides to the surfaces of the plurality of binding particles comprising a poly-cationic substance, wherein the sample has a volume from 0.5 microliters to 3 milliliters;
wherein the first set of conditions includes a first pH of about 8.5 or less and a first temperature, wherein the first temperature is between about 30° C. and about 50° C.;
releasing the polynucleotide from the plurality of binding particles under a second set of conditions; and
wherein the second set of conditions includes increasing the pH to a second pH by addition of a hydroxide solution and increasing the temperature to a second temperature, wherein the second temperature is between about 80° C. and about 100° C.
2. The method of claim 1 , wherein the second pH is about 11.3 or greater.
3. The method of claim 1 , wherein the poly-cationic substance is covalently bound to the surfaces of the binding particles.
4. The method of claim 1 , wherein the binding particles comprise one or more carboxylic groups to provide an attachment point for the poly-cationic substance.
5. The method of claim 4 , further comprising treating magnetic beads with NHS and MAC to form the binding particles.
6. The method of claim 1 , wherein the heating step comprises heating the plurality of binding particles in the presence of a liquid, wherein the second temperature is insufficient to boil the liquid.
7. The method of claim 1 , further comprising maintaining the second temperature for between about 1 and 10 minutes.
8. The method of claim 1 , wherein the sample is heated for about 15 minutes or less while contacting the binding particles with the basic solution thereby releasing the polynucleotides.
9. The method of claim 1 , wherein the poly-cationic substance has a molecular weight of less than about 30,000 Da.
10. The method of claim 1 , wherein the poly-cationic substance has a molecular weight of less than about 800 Da.
11. The method of claim 1 , wherein the ratio of the volume of the sample in the process chamber to the volume of liquid into which the polynucleotides are released is at least about 10.
12. The method of claim 1 , wherein the particles occupy about 75 percent or less of the total volume of the process chamber.
13. A method for processing a polynucleotide-containing sample, the method comprising:
retaining polynucleotides from a sample on a plurality of binding particles in a process chamber under a first set of conditions, wherein the retaining step comprises binding the polynucleotides to the surfaces of the plurality of binding particles comprising a poly-cationic substance; wherein the sample has a volume from 0.5 microliters to 3 milliliters;
wherein the first set of conditions includes a first pH of about 8.5 or less and a first temperature, wherein the first temperature is about 60° C.;
releasing the polynucleotide from the plurality of binding particles under a second set of conditions; and
wherein the second set of conditions includes increasing the pH to a second pH by addition of a hydroxide solution and increasing the temperature to a second temperature, wherein the second temperature is between about 80° C. and about 100° C.
14. The method of claim 13 , wherein the second pH is about 11.3 or greater.
15. The method of claim 13 , wherein the poly-cationic substance is covalently bound to the surfaces of the binding particles.
16. The method of claim 13 , wherein the binding particles comprise one or more carboxylic groups to provide an attachment point for the poly-cationic substance.
17. The method of claim 16 , further comprising treating magnetic particles with NHS and EDAC to form the binding particles.
18. The method of claim 13 , further comprising maintaining the second temperature for between about 1 and 10 minutes.
19. The method of claim 13 , wherein the sample is heated for about 15 minutes or less while contacting the binding particles with the basic solution thereby releasing the polynucleotides.
20. The method of claim 13 , wherein the poly-cationic substance has a molecular weight of less than about 30,000 Da.
21. The method of claim 13 , wherein the poly-cationic substance has a molecular weight of less than about 800 Da.