Control of chemical reactions using isotachophoresis

Patent metadata

Field	Value
Publication number	US-10073054-B2
Application number	US-201615081415-A
Country	US
Kind code	B2
Filing date	Mar 25, 2016
Priority date	Oct 7, 2008
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

Isotachophoresis (ITP) is exploited to control various aspects of chemical reactions. In a first aspect, at least one of the reactants of a chemical reaction is confined to an ITP zone, but the resulting product of the chemical reaction is separated from this ITP zone by the ITP process. In a second aspect, one or more reactants of a chemical reaction are confined to an ITP zone, and one or more other reactants of the chemical reaction are not confined to this ITP zone. In a third aspect, ITP is employed to confine at least one reactant of a chemical reaction to an ITP zone, and at least one reactant of the chemical reaction is delivered to the ITP zone in two or more discrete doses. These aspects are especially relevant to performing polymerase chain reactions using chemical denaturants as opposed to thermal cycling.

First claim

The invention claimed is:

1. A method of performing a chemical reaction, the method comprising:
providing at least a first reactant in a liquid flow channel;
providing at least one catalyst, enzyme, or denaturant in said liquid flow channel;
allowing said first reactant to chemically react in said liquid flow channel with said catalyst, enzyme, or denaturant to produce at least a first product, wherein said catalyst, enzyme, or denaturant is not consumed during production of said first product; and
confining said first product by isotachophoresis (ITP) to a first ITP zone in said liquid flow channel.

2. The method of claim 1 , wherein said first reactant is not confined to said first ITP zone.

3. The method of claim 1 , wherein said first reactant is confined to said first ITP zone.

4. The method of claim 1 , wherein providing said catalyst, enzyme, or denaturant in said liquid flow channel comprises providing said catalyst, enzyme, or denaturant in a reservoir fluidly coupled to said liquid flow channel and moving said catalyst, enzyme, or denaturant from said reservoir to said liquid flow channel.

5. The method of claim 1 , wherein said catalyst, enzyme, or denaturant is an enzyme.

6. The method of claim 5 , wherein said enzyme is a polymerase.

7. The method of claim 6 , wherein said polymerase is a thermostable DNA polymerase, a heat-labile polymerase, a non-thermostable polymerase, or a polymerase fragment.

8. The method of claim 6 , wherein said polymerase comprises a Klenow fragment of DNA polymerase I or a Taq DNA polymerase.

9. The method of claim 1 , wherein said catalyst, enzyme, or denaturant is a denaturant.

10. The method of claim 9 , wherein said denaturant comprises urea or formamide.

11. The method of claim 1 , wherein said first ITP zone is substantially stationary in said liquid flow channel.

12. The method of claim 1 , wherein said first reactant is substantially stationary in said liquid flow channel.

13. The method of claim 1 , wherein said first reactant has an ITP electromigration mobility direction and said at least one catalyst, enzyme, or denaturant flows towards said first reactant in a direction opposite to said ITP electromigration mobility direction of said first reactant.

14. The method of claim 13 , wherein said at least one catalyst, enzyme, or denaturant further flows past said first reactant in said liquid flow channel.

15. The method of claim 13 , wherein flow of said at least one catalyst, enzyme, or denaturant is driven by pressure.

16. The method of claim 1 , wherein said first reactant has an ITP electromigration mobility direction and said at least one catalyst, enzyme, or denaturant electromigrates towards said first reactant in a direction opposite to said ITP electromigration mobility direction of said first reactant.

17. The method of claim 16 , wherein said at least one catalyst, enzyme, or denaturant further electromigrates past said first reactant in said liquid flow channel.

18. The method of claim 1 , wherein said first reactant comprises a nucleic acid.

19. The method of claim 1 , wherein said first reactant comprises a deoxyribonucleic acid.

20. The method of claim 19 , wherein said deoxyribonucleic acid is double-stranded.

21. The method of claim 1 , wherein said first reactant comprises a ribonucleic acid.

22. The method of claim 1 , wherein said first reactant comprises at least one primer, oligonucleotide, or dNTP.

23. The method of claim 22 , wherein said catalyst, enzyme, or denaturant is a polymerase and wherein said chemical reaction of said first reactant with said polymerase is a primer extension reaction.

24. The method of claim 1 , wherein said first product comprises a deoxyribonucleic acid.

25. The method of claim 24 , wherein said first product is single-stranded.

26. The method of claim 24 , wherein said first product is double-stranded.

27. The method of claim 1 , wherein said first product comprises a ribonucleic acid.

28. The method of claim 1 , further comprising fluorescently labeling at least one of said first reactant or said first product.

29. The method of claim 28 , wherein said fluorescent labeling comprises labeling with a fluorescent dye, an intercalating fluorescent dye, a sequence specific fluorescent probe, or a molecular beacon.

30. The method of claim 28 , where said fluorescent labeling comprises labeling with SYBR Green I, SYTO 13, or SYBR Green II.

31. The method of claim 28 , further comprising detecting fluorescence of at least one of said fluorescently-labeled first reactant or said fluorescently-labeled first product.

32. The method of claim 1 , further comprising detecting said first product.

33. The method of claim 1 , further comprising monitoring said first product in real-time.

34. The method of claim 1 , wherein said chemical reaction of said first reactant with said catalyst, enzyme, or denaturant comprises at least a part of a polymerase chain reaction.

35. The method of claim 1 , wherein said chemical reaction of said first reactant with said catalyst, enzyme, or denaturant is performed at constant temperature.

36. The method of claim 1 , wherein said chemical reaction of said first reactant with said catalyst, enzyme, or denaturant is performed at one or more temperatures that are elevated above an ambient temperature.

37. The method of claim 1 , further comprising providing a second reactant wherein said second reactant comprises nucleic acid or dNTP.

38. The method of claim 37 , wherein said first reactant comprises nucleic acid.

39. A method of performing a chemical reaction, the method comprising:
providing a first nucleic acid reactant;
providing a second nucleic acid reactant, wherein at least one of said first nucleic acid reactant and said second nucleic acid reactant is confined by isotachophoresis (ITP) to a first ITP zone in a liquid flow channel; and
allowing said first nucleic acid reactant and said second nucleic acid reactant to react in said first ITP zone to produce at least a first product.

40. A method of performing a chemical reaction, the method comprising:
providing a first reactant, wherein said first reactant is confined by isotachophoresis (ITP) to a first ITP zone in a liquid flow channel;
providing a catalyst, enzyme, or denaturant in said first ITP zone; and
allowing said first reactant to react in said first ITP zone to produce at least a first product, wherein said catalyst, enzyme, or denaturant facilitates production of said first product and wherein said catalyst, enzyme, or denaturant is not consumed during production of said first product.

Assignees

• Univ Leland Stanford Junior

Inventors

• Santiago Juan G
• Persat Alexandre

Classifications

• G01N27/447

  using electrophoresis · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• G01N27/44795

  Isoelectric focusing · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• C12M1/42

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