Methods for improved DNA release from binding substrates and/or decreasing PCR inhibition in pathogen detection
Disclosed herein are processes for collecting nucleic acids from particulate samples. One embodiment disclosed herein relates to the use of ultrasonic energy to simultaneously shear large nucleic acid molecules and large particulates to very small sizes prior to or during a chemical binding step to a nucleic acid binding surface. Another embodiment involves crushing the nucleic acid binding surface prior to eluting the bound nucleic acid molecules to enable better wetting of the nucleic acid binding surface and easier diffusion of bound nucleic acid molecules out of the nucleic acid binding surface.
1. A process for collecting nucleic acids from a particulate sample comprising:
(a) mixing an aliquot of a particulate sample containing nucleic acids with a nucleic acid binding solution;
(b) shearing the nucleic acids in the mixture of step (a) by ultrasound;
(c) transferring the mixture of step (b) into a device comprising a chamber interior comprising a fibrous nucleic acid binding surface, the chamber interior being capable of expanding in size in at least one dimension; the fibers of the fibrous nucleic acid binding surface expanding to at least partially fill the chamber interior upon wetting with the mixture;
(d) expelling the mixture from the device by compression of the fibrous nucleic acid binding surface while retaining the fibrous nucleic acid binding surface in the chamber interior;
(e) powderizing the fibrous nucleic acid binding surface;
(f) contacting the powderized nucleic acid binding surface with an elution buffer; and
(g) separating the elution buffer from the powderized nucleic acid binding surface, whereby the elution buffer contains nucleic acids from the particulate sample.
2. The process of claim 1 comprising after step (d) and before step (e) the further steps of:
washing the fibrous nucleic acid binding surface with a wash solution; and
expelling the wash solution by compression of the fibrous nucleic acid binding surface while retaining the fibrous nucleic acid binding surface in the chamber interior.
3. The process of claim 2 , wherein the expelling is performed by vacuum.
4. The process of claim 1 , wherein the expelling of step (d) is performed by vacuum.
5. The process of claim 1 , wherein the fibrous nucleic acid binding surface is non-magnetic.
6. The process of any of claims 1 - 5 , wherein the fibrous nucleic acid binding surface is a clean silica surface.
7. The process of claim 6 , wherein the clean silica surface is a clean, activated silica surface.
8. The process of claim 7 , wherein the clean, activated silica surface comprises silica wool.
9. The process of any of claims 1 - 5 , wherein the nucleic acid binding solution comprises a chaotropic salt.
10. The process of any of claims 1 - 5 , wherein the particulate sample comprises eukaryotic cells, prokaryotic cells, viruses, and a combination thereof.
11. The process of any of claims 1 - 5 , wherein the particulate sample is a food sample or a clinical sample.
12. A process for collecting nucleic acids from a particulate sample comprising:
(a) transferring an aliquot of a particulate sample containing nucleic acids into a device comprising
(i) a chamber interior comprising a non-magnetic fibrous nucleic acid binding surface, the chamber interior being capable of expanding in size in at least one dimension; and
(ii) a nucleic acid binding solution; the fibers of the non-magnetic fibrous nucleic acid binding surface expanding to at least partially fill the chamber interior upon wetting with the particulate sample and the nucleic acid binding solution;
(b) shearing the nucleic acids in the sample by ultrasound;
(c) mixing the particulate sample with the nucleic acid binding solution;
(d) expelling the nucleic acid binding solution from the device by compression of the non-magnetic fibrous nucleic acid binding surface while retaining the non-magnetic fibrous nucleic acid binding surface in the chamber interior;
(e) removing the non-magnetic fibrous nucleic acid binding surface from the device;
(f) powderizing the non-magnetic fibrous nucleic acid binding surface
(g) contacting the powderized nucleic acid binding surface with an elution buffer; and
(h) separating the elution buffer from the powderized non-magnetic nucleic acid binding surface, whereby the elution buffer contains nucleic acids from the particulate sample.
13. A process for collecting nucleic acids from a particulate sample comprising:
(a) mixing an aliquot of a particulate sample containing nucleic acids with a nucleic acid binding solution;
(b) shearing the nucleic acids in the mixture of step (a) by ultrasound;
(c) transferring the mixture of step (b) into a device comprising a chamber interior comprising a fibrous nucleic acid binding surface, the chamber interior being capable of expanding in size in at least one dimension; the fibers of the fibrous nucleic acid binding surface expanding to at least partially fill the chamber interior upon wetting with the mixture;
(d) expelling the mixture from the device by compression of the fibrous nucleic acid binding surface while retaining the fibrous nucleic acid binding surface in the chamber interior;
(e) powderizing the fibrous nucleic acid binding surface;
(f) contacting the powderized nucleic acid binding surface with an elution buffer; and
(g) separating the elution buffer from the powderized nucleic acid binding surface, whereby the elution buffer contains nucleic acids from the particulate sample.
14. The process of claim 13 , wherein the fibrous nucleic acid binding surface is non-magnetic.
15. The process of claim 13 comprising after step (d) and before step (e) the further step of removing the fibrous nucleic acid binding surface from the device.
16. A process for collecting nucleic acids from a particulate sample comprising:
(a) transferring an aliquot of a particulate sample containing nucleic acids into a device comprising
(i) a chamber interior comprising a fibrous nucleic acid binding surface, the chamber interior being capable of expanding in size in at least one dimension; and
(ii) a nucleic acid binding solution;
the fibers of the fibrous nucleic acid binding surface expanding to at least partially fill the chamber interior upon wetting with the particulate sample and the nucleic acid binding solution;
(b) shearing the nucleic acids in the sample by ultrasound;
(c) mixing the particulate sample with the nucleic acid binding solution;
(d) expelling the nucleic acid binding solution from the device by compression of the fibrous nucleic acid binding surface while retaining the fibrous nucleic acid binding surface in the chamber interior;
(e) powderizing the fibrous nucleic acid binding surface;
(f) contacting the powderized nucleic acid binding surface with an elution buffer; and
(g) separating the elution buffer from the powderized nucleic acid binding surface, whereby the elution buffer contains nucleic acids from the particulate sample.
17. The process of claim 16 , wherein the fibrous nucleic acid binding surface is non-magnetic.
18. The process of claim 16 comprising after step (d) and before step (e) the further step of removing the fibrous nucleic acid binding surface from the device.
19. The process of claim 1 comprising after step (d) and before step (e) the further steps of:
washing the fibrous nucleic acid binding surface with a wash solution; expelling the wash solution by compression of the fibrous nucleic acid binding surface while retaining the fibrous nucleic acid binding surface in the chamber interior; and
removing the fibrous nucleic acid binding surface from the device.