METHOD, SUBSTRATE AND DEVICE FOR SEPARATING NUCLEIC ACIDS

• US 20160002621A1
• Filed: 07/01/2014
• 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, Moore, David Roger, Duthie, Robert Scott, Misner, Matthew Jeremiah, Grossmann, Gregory Andrew, Dees, Elizabeth Marie, Spooner, Patrick McCoy, Kvam, Erik Leeming, Burns, Andrew Arthur Paul, Watkins, Vicki Herzl
• Granted Patent: US 10,472,620 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
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates

Abstract

A method is provided herein, the method includes: applying a sample comprising target nucleic acids to a sample application zone of a substrate; and flowing a nucleic acid amplification reaction mixture across a length of the substrate through the sample application zone to amplify the target nucleic acid forming a nucleic acid amplification product; wherein the target nucleic acid having a first molecular weight is substantially immobilized at the sample application zone and wherein the amplification product having a second molecular weight migrates away from the sample application zone. An associated device is also provided.

Claims

1. A method comprising:

applying a sample comprising target nucleic acids to a sample application zone of a substrate; and

flowing a nucleic acid amplification reaction mixture across a length of the substrate through the sample application zone to amplify the target nucleic acid forming a nucleic acid amplification product;

wherein the target nucleic acid having a first molecular weight is substantially immobilized at the sample application zone and the amplification product having a second molecular weight migrates away from the sample application zone.

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

3. The method of claim 2, wherein the sample comprises the lysis reagent.

4. The method of claim 2, wherein the lysis reagent is impregnated in the sample application zone.

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

6. The method of claim 5, wherein the migration modifier comprises a chaotrope.

7. The method of claim 5, wherein the migration modifier comprises guanidinium thiocyanate.

8. The method of claim 1, wherein flowing the nucleic acid amplification reaction mixture separates the target nucleic acids and the amplification product according to their molecular weights.

9. The method of claim 1, wherein flowing the nucleic acid amplification reaction mixture washes the lysis reagent from the substrate.

10. The method of claim 1, wherein flowing the nucleic acid amplification reaction mixture washes away one or more inhibitors present on the sample application zone.

11. The method of claim 1, wherein the amplification products migrate by lateral flow.

12. The method of claim 1, further comprising applying a flow bather to prevent the migration of the amplification products until the barrier is removed.

13. The method of claim 1, wherein a first amplification reaction occurs at the sample application zone to generate a first amplification product.

14. The method of claim 13, wherein one or more amplification reactions occur during migration of the first amplification product.

15. The method of claim 1, wherein the substrate comprises one or more detection probes located at the sample application zone, downstream of the sample application zone or combinations thereof.

16. The method of claim 15, wherein the detection probes are impregnated in the substrate under dried condition, added as part of the amplification reaction mixture, added as a solution or combinations thereof.

17. The method of claim 15, further comprising detecting the amplification product using the detection probes.

18. The method of claim 1, wherein the amplification product is immobilized on the substrate by a physical interaction with the substrate, using a capturing probe, using a detection probe or combinations thereof.

19. The method of claim 18, wherein the amplification product is captured on the substrate by hybridization with a capturing probe physically bound to the substrate to form captured amplification product.

20. The method of claim 19, further comprising:

flowing a solution comprising primary detection probes along the length of the substrate to bind to the captured amplification product to form a primary detection probe bound amplification product.

21. 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 bound amplification product.

22. The method of claim 1, wherein the first molecular weight is in a range of at least about 50 kb.

23. The method of claim 1, wherein the first molecular weight is in a range of from about 50 kb to about 150 kb.

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

25. The method of claim 24, wherein the substrate further comprises a detection zone near the second end.

26. The method of claim 25, wherein the nucleic acids having a second molecular weight are substantially positioned around the detection zone.

27. The method of claim 24, further comprising:

providing a wicking pad adjacent to the second end.

28. The method of claim 24, further comprising:

providing a stopping pad adjacent to the second end.

29. The method of claim 1, further comprising:

flowing a washing buffer through the sample application zone along the length of the substrate.

30. The method of claim 1, wherein the nucleic acids are DNA, RNA or a combination thereof.

31. The method of claim 1, wherein the substrate is made of 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 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.

32. A method comprising:

applying a sample comprising target nucleic acids 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 amplify the target nucleic acid forming a nucleic acid amplification product; and

separating the target nucleic acids from the amplified nucleic acids, wherein the target nucleic acid having a first molecular weight is substantially immobilized at the sample application zone and the amplification product having a second molecular weight migrates away from the sample application zone;

wherein the target nucleic acids are amplified, and separated without a washing step.

33. A substrate, comprising:

a sample application zone for applying a sample comprising a target nucleic acid having a first molecular weight and flowing an amplification reaction mixture along the length of the substrate through the sample application zone forming a nucleic acid amplification product having a second molecular weight; and

a detection zone for separating the nucleic acid amplification product having second molecular weight from the target nucleic acids having first molecular weight according to molecular weights of the amplified nucleic acids and target nucleic acids.

34. The substrate of claim 33, wherein the sample application zone comprises a lysis reagent.

35. The substrate of claim 33, wherein the sample application zone further comprises a migration modifier.

36. The substrate of claim 33, wherein the migration modifier comprises a chaotrope.

37. The substrate of claim 33 further comprising a flow barrier to immobilize the amplification product.

38. The substrate of claim 33 further comprising one or more detection probes disposed on the substrate downstream of the sample application zone.

39. The substrate of claim 33, further comprising:

a stopping pad adjacent to an opposite end of the sample application zone.

40. The substrate of claim 33 is an elongated strip comprising a first end comprising the sample application zone, and a second end comprising the detection zone.

41. The substrate of claim 33, wherein the substrate comprises cellulose, nitrocellulose, modified porous nitrocellulose or cellulose based substrates, polyethyleneglycol-modified nitrocellulose, a cellulose acetate membrane, a nitrocellulose mixed ester membrane, a glass 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, glass, wood, metal, or a combination thereof.

42. The substrate of claim 33 is operatively coupled to an amplification reagent reservoir, an amplification reagent wicking pad, a wash reagent reservoir, a wash reagent wicking pad and combinations thereof.

43. A device, comprising:

a substrate, comprising;

a sample application zone for applying a target nucleic acid sample and flowing an amplification reaction mixture along the length of the substrate through the sample application zone forming a nucleic acid amplification product; and

a detection zone for separating the nucleic acid amplification product from the target nucleic acids according to molecular weights of the amplified nucleic acids and target nucleic acids; and

an amplification reagent reservoir.

44. The device of claim 43 further comprising an amplification reagent wicking pad.

45. The device of claim 43 further comprising an amplification zone heating unit.

46. The device of claim 43 further comprising a heating unit for drying the sample at the sample application zone.