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) 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) mixing the particulate sample with the nucleic acid binding solution;
(c) 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;
(d) powderizing the fibrous nucleic acid binding surface;
(e) contacting an elution buffer with the powderized nucleic acid binding surface; and
(f) 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 further comprising after step (c) and before step (d) 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 (c) 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. The process of any of claims 1 - 5 further comprising after step (c) and before step (d) the further step of removing the fibrous nucleic acid binding surface from the device.
13. A process for collecting nucleic acids from a particulate sample comprising:
(a) exposing an aliquot of a particulate sample containing nucleic acids to a nucleic acid binding surface in the presence of a nucleic acid binding solution, said nucleic acid binding surface comprising a fibrous material capable of expansion upon wetting;
(b) binding the nucleic acids to the nucleic acid binding surface by expanding the fibrous material;
(c) separating the particulate sample from the nucleic acid binding surface comprising nucleic acids bound thereto by compression of the fibrous material;
(d) washing the nucleic acid binding surface with a wash solution whereby the fibrous material is again expanded;
(e) separating the wash solution from the nucleic acid binding surface comprising nucleic acids bound thereto by compression of the fibrous material;
(f) powderizing the nucleic acid binding surface;
(g) exposing an aliquot of elution buffer to the powderized nucleic acid binding surface; and
(h) eluting nucleic acids from the powderized nucleic acid binding surface.
14. The process of claim 13 , wherein the fibrous nucleic acid binding surface is non-magnetic.