Method for processing polynucleotide-containing samples
View Patent ↗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 separating one or more polynucleotides from a sample containing polymerase chain reaction inhibitors, the method comprising:
contacting a solution of the sample with a plurality of polynucleotide binding particles, wherein the binding particles are configured to preferentially retain the one or more polynucleotides in the sample as compared to polymerase chain reaction inhibitors;
wherein the sample solution has a volume from 0.5 microliters to 3 milliliters;
wherein the plurality of binding particles have a volume less than 5 microliters, and surfaces that comprise a polycationic polyamide configured to bind polynucleotides in preference to polymerase chain reaction inhibitors at a pH of 8.5 or less;
removing the solution containing inhibitors from the plurality of binding particles; and
releasing the one or more polynucleotides from the binding particles into a single volume of liquid wherein the ratio of the volume of sample solution to the volume of liquid into which the polynucleotides are released is between 50:1 and 1000:1, wherein the releasing occurs at a pH of 11.4 or greater.
2. The method of claim 1 , wherein the one or more polynucleotides has a size of less than 7.5 Mbp.
3. The method of claim 1 , wherein contacting the sample with the plurality of polynucleotide binding particles comprises actuating a thermally actuated pressure source to apply a pressure to the sample.
4. The method of claim 3 , wherein contacting the sample with the plurality of polynucleotide binding particles comprises opening a thermally actuated valve to place the sample in fluid communication with the binding particles.
5. The method of claim 1 , additionally comprising, prior to the releasing step, washing the particles with a volume of wash solution less than 50 microliters.
6. The method of claim 5 wherein the wash solution comprises a detergent.
7. The method of claim 1 , wherein the polymerase chain reaction inhibitors comprise at least one of hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds, DNA binding proteins, or a saccharide.
8. The method of claim 1 , wherein the polycationic polyamide is polyethyleneimine.
9. The method of claim 8 , wherein the polyethyleneimine has a molecular weight in the range 600-800 Da.
10. The method of claim 1 , wherein the polycationic polyamide is selected from the group consisting of poly-DL-ornithine, poly-L-lysine, and poly-D-lysine.
11. The method of claim 1 , wherein the releasing comprises heating the plurality of binding particles to a temperature of between about 50° C. and about 100° C.
12. The method of claim 11 , wherein the plurality of binding particles is heated in the presence of a liquid and the temperature is insufficient to boil the liquid in the presence of the plurality of binding particles during heating.
13. The method of claim 11 , wherein the temperature is maintained for between about 1 and 10 minutes.
14. The method of claim 1 , wherein the method does not comprise centrifugation of the binding particles.
15. The method of claim 1 , wherein the time required for completing the contacting, concentrating, and releasing steps is less than 15 minutes.
16. The method of claim 1 , wherein the sample has a volume larger than the volume of the polynucleotide binding particles having the one or more polynucleotides bound thereto by a factor of at least about 10.
17. The method of claim 1 , wherein the polycationic polyamide is covalently bound to the surfaces of the binding particles.
18. The method of claim 1 , wherein the polycationic polyamide is poly-L-lysine or poly-D-lysine and has an average molecular weight of between about 7,500 Da and about 35,000 Da.
19. The method of claim 1 , wherein the polycationic polyamide is poly-L-lysine or poly-D-lysine and has a median molecular weight of between about 15,000 Da and about 25,000 Da.
20. The method of claim 1 , wherein the binding particles are made of a polymeric material selected from the group consisting of: polystyrene, latex polymers, polyacrylamide, and polyethylene oxide.
21. The method of claim 20 , wherein the polymeric material is modified to provide one or more carboxylic groups and/or one or more amino groups, wherein the groups provide an attachment point for one or more ligands.
22. The method of claim 1 , wherein the binding particles have an average diameter of between about 4 microns and about 20 microns.
23. The method of claim 1 , wherein the binding particles are present in a density of about 10 8 particles per milliliter.
24. The method of claim 1 , wherein at least some of the binding particles are magnetic.
25. The method of claim 1 , wherein the polycationic polyamide is resistant to pronase degradation.
26. The method of claim 1 , wherein the contacting takes place in the presence of lysis reagents.
27. The method of claim 26 , wherein the contacting in the presence of lysis reagents comprises heating the sample to a temperature more than 50° C.
28. The method of claim 26 , wherein the contacting in the presence of lysis reagents takes place for a period of time between 5 to 15 minutes.