METHODS FOR CAPTURING NUCLEIC ACIDS

• US 20160002622A1
• Filed: 12/11/2014
• Published: 01/07/2016
• Est. Priority Date: 07/01/2014
• Status: Active Grant

• Current Assignee: Global Life Sciences Solutions Operations UK Ltd. (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Nelson, John Richard, Li, Bing
• Granted Patent: US 10,870,845 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers

Abstract

A method is provided herein, wherein the method of capturing a target nucleic acid, comprises applying a nucleic acid capture probe to a capture zone of a needs definition, wherein the nucleic acid capture probe having a first molecular weight comprises at least a sequence that is complimentary to at least a portion of the target nucleic acid sequence and the nucleic acid capture probe is substantially immobilized at the capture zone of the substrate. The method further comprises applying a sample comprising the target nucleic acid having a second molecular weight to a sample application zone of the substrate; wherein the sample comprising the target nucleic acid flows across a length of the substrate from the sample application zone to the capture zone by lateral flow, and the target nucleic acid is captured by the nucleic acid capture probes by hybridization to the capture zone.

Claims

1. A method of capturing a target nucleic acid, comprising:

applying a nucleic acid capture probe to a capture zone of a substrate, wherein the nucleic acid capture probe having a first molecular weight comprises at least a sequence that is complimentary to at least a portion of the target nucleic acid sequence and the nucleic acid capture probe is substantially immobilized at the capture zone of the substrate;

applying a sample comprising the target nucleic acid having a second molecular weight to a sample application zone of the substrate;

wherein the sample comprising the target nucleic acid flows across a length of the substrate from the sample application zone to the capture zone by lateral flow, and the target nucleic acid is captured by the nucleic acid capture probes by hybridization at the capture zone,

wherein the nucleic acid capture probe is a rolling circle amplification (RCA) product.

2. The method of claim 1, further comprising flowing a liquid across the length of the substrate from the sample application zone to the capture zone, wherein the target nucleic acids migrate away from the sample application zone to the capture zone by lateral flow.

3. The method of claim 1, wherein the first molecular weight of the nucleic acid capture probes is in a range from about 20,000 bases to about 150,000 bases.

4. The method of claim 1, wherein the nucleic acid capture probe comprises multiple tandem repeat sequences.

5. The method of claim 1, wherein the nucleic acid capture probe comprises 20 to 150 nucleotides of tandem repeat sequences.

6. The method of claim 1, wherein the nucleic acid capture probe comprises single stranded nucleic acids.

7. The method of claim 1, wherein the nucleic acid capture probe comprises deoxyribonucleic acids (DNA), ribonucleic acids (RNA), nucleotide analogues, or combinations thereof.

8. The method of claim 1, wherein the nucleic acid capture probe is immobilized on the substrate by entanglement with the substrate.

9. The method of claim 1, wherein the sample application zone and the capture zone are same.

10. The method of claim 1, further comprising contacting the sample with a lysis reagent.

11. The method of claim 10, wherein the sample comprises the lysis reagent.

12. The method of claim 10, wherein the lysis reagent is impregnated in the sample application zone.

13. The method of claim 1, wherein the sample application zone further comprises a migration modifier.

14. The method of claim 13, wherein the migration modifier comprises a chaotrope.

15. The method of claim 13, wherein the migration modifier comprises guanidinium thiocyanate.

16. The method of claim 1, wherein the sample further comprises template nucleic acid having a third molecular weight and the liquid is a nucleic acid amplification reaction mixture that amplifies the template nucleic acids to form the target nucleic acid amplification product.

17. The method of claim 16, wherein one or more amplification reactions occur at the sample application zone or during migration of the target nucleic acid amplification product.

18. The method of claim 1, further comprising washing the substrate to remove one or more inhibitors, non-target nucleic acids, the lysis reagents or combinations thereof present on the substrate.

19. The method of claim 1, wherein a primary detection probe is added to the target nucleic acid, the nucleic acid capture probe, or combinations thereof.

20. The method of claim 1, wherein one or more of the target nucleic acid, the nucleic acid capture probe comprises a primary detection probe.

21. The method of claim 20, wherein the primary detection probe comprises a chromophore moiety, a fluorescent moiety, a phosphorescence moiety, an affinity probe, a magnetic probe, a paramagnetic probe, a metallic probe or combinations thereof.

22. The method of claim 20, wherein the primary detection probe comprises a binding moiety selected from an antibody, a biotin, a streptavidin bead, a gold particle or combinations thereof.

23. The method of claim 20, further comprising:

flowing a solution comprising secondary detection probes along the length of the substrate to bind to the primary detection probe, target nucleic acid, nucleic acid capture probe or combinations thereof.

24. The method of claim 1, wherein the substrate is an elongated strip comprising a first end, a sample application zone, a second end and a capture zone.

25. The method of claim 24, wherein the substrate further comprises a wicking pad adjacent to the second end.

26. The method of claim 24, wherein the substrate further comprises a stopping pad adjacent to the second end.

27. The method of claim 1, wherein the substrate comprises cellulose membrane, a nitrocellulose membrane, modified porous nitrocellulose or cellulose based substrates, polyethyleneglycol-modified nitrocellulose, a cellulose acetate membrane, a nitrocellulose mixed ester membrane, a glass fiber, a quartz fiber, a polyethersulfone membrane, a nylon membrane, a polyolefin membrane, a polyester membrane, a polycarbonate membrane, a polypropylene membrane, a polyvinylidene difluoride membrane, a polyethylene membrane, a polystyrene membrane, a polyurethane membrane, a polyphenylene oxide membrane, a poly(tetrafluoroethylene-co-hexafluoropropylene) membrane, or a combination thereof.

28. A method of capturing target nucleic acids, comprising:

applying nucleic acid capture probes to a capture zone of a substrate, wherein the nucleic acid capture probes having a first molecular weight comprise at least a sequence that is complementary to at least a portion of the target nucleic acid sequence and the nucleic acid capture probes are substantially immobilized at the capture zone of the substrate;

applying a sample comprising the target nucleic acids having a second molecular weight to a sample application zone of the substrate; and

flowing a liquid across a length of the substrate from the sample application zone to the capture zone, wherein the target nucleic acids migrate away from the sample application zone to the capture zone by lateral flow and are captured by the nucleic acid capture probes by hybridization.

29. A method of capturing target nucleic acids, comprising:

applying a nucleic acid capture probe to a capture zone of a substrate, wherein the nucleic acid capture probe having a first molecular weight comprises at least a sequence that is complementary to at least a portion of the target nucleic acid sequence and the nucleic acid capture probe is substantially immobilized at the capture zone of the substrate;

applying a sample comprising one or more of the target nucleic acid having a second molecular weight, a template nucleic acid having a third molecular weight to a sample application zone of a substrate;

flowing a nucleic acid amplification reaction mixture across a length of the substrate through the sample application zone to the capture zone, wherein the template nucleic acids are amplified to form the target nucleic acids;

separating the target nucleic acids from the template nucleic acids by lateral flow,

wherein the target nucleic acids migrate away from the sample application zone to the capture zone and are captured by the nucleic acid capture probes by hybridization without a washing step.