Control of chemical reactions using isotachophoresis

Patent metadata

Field	Value
Publication number	US-10830732-B2
Application number	US-201816121219-A
Country	US
Kind code	B2
Filing date	Sep 4, 2018
Priority date	Oct 7, 2008
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

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. An isotachophoresis (ITP) apparatus comprising:
at least a first liquid flow channel containing:
a) a leading electrolyte;
b) a trailing electrolyte;
c) a catalyst, an enzyme, or a denaturant; and
d) a nucleic acid;

wherein said first liquid flow channel is in liquid communication with at least a first reservoir, a second reservoir, or a third reservoir, wherein:
i) if present, said first reservoir comprises said leading electrolyte;
ii) if present, said second reservoir comprises said trailing electrolyte; or
iii) if present, said third reservoir comprises said catalyst, enzyme, or denaturant.

2. The apparatus of claim 1 , wherein said first liquid flow channel comprises a second liquid flow channel connected to a third liquid flow channel wherein said second liquid flow channel comprises said leading electrolyte and said third liquid flow channel comprises said trailing electrolyte.

3. The apparatus of claim 2 , further comprising a reservoir fluidly coupled to said first, second, or third liquid flow channels.

4. The apparatus of claim 1 , wherein said nucleic acid comprises a deoxyribonucleic acid.

5. The apparatus of claim 4 , wherein said deoxyribonucleic acid is double-stranded.

6. The apparatus of claim 4 , wherein said deoxyribonucleic acid is single-stranded.

7. The apparatus of claim 1 , wherein said first liquid flow channel further comprises a ribonucleic acid.

8. The apparatus of claim 1 , wherein said first liquid flow channel further comprises at least one primer, oligonucleotide, or dNTP.

9. The apparatus of claim 1 , wherein said first liquid flow channel further comprises a confined nucleic acid template.

10. The apparatus of claim 1 , wherein said first liquid flow channel further comprises a confined oligonucleotide.

11. The apparatus of claim 1 , wherein said first liquid flow channel further comprises an electrophoretic spacer ion.

12. The apparatus of claim 1 , wherein said first liquid flow channel further comprises a fluorescent dye, an intercalating fluorescent dye, a sequence specific fluorescent probe, or a molecular beacon.

13. The apparatus of claim 1 , wherein said first liquid flow channel further comprises SYBR Green I, SYTO 13, or SYBR Green II.

14. The apparatus of claim 1 , wherein said catalyst, enzyme, or denaturant comprises a denaturant.

15. The apparatus of claim 14 , wherein said denaturant comprises a nucleic acid denaturant.

16. The apparatus of claim 1 , wherein said catalyst, enzyme, or denaturant comprises an enzyme.

17. The apparatus of claim 16 , wherein said enzyme is a polymerase.

18. The apparatus of claim 17 , wherein said polymerase is a thermostable DNA polymerase, a heat-labile polymerase, a non-thermostable polymerase, or a polymerase fragment.

19. The apparatus of claim 17 , wherein said polymerase comprises a Klenow fragment of DNA polymerase I or a Taq DNA polymerase.

20. The apparatus of claim 1 , wherein said apparatus is configured to be coupled to a system for monitoring a concentration of at least one of a reactant or a product in said first liquid flow channel.

21. The apparatus of claim 1 , wherein said apparatus is coupled to a fluorescence detector.

22. The apparatus of claim 1 , wherein said apparatus is coupled to a heater.

23. The apparatus of claim 1 , wherein said apparatus is coupled to a temperature controller.

24. The apparatus of claim 1 , wherein said reservoir is a first reservoir comprising said first reservoir and said second reservoir are both present, wherein said first reservoir comprises said leading electrolyte, and wherein said second reservoir comprises said trailing electrolyte.

25. The apparatus of claim 24 , wherein said third reservoir is present and wherein said third reservoir comprises said catalyst, enzyme, or denaturant.

26. An isotachophoresis (ITP) apparatus comprising:
at least a first liquid flow channel containing:
a) a leading electrolyte,
b) a trailing electrolyte,
c) a catalyst, an enzyme, or a denaturant, and
d) a nucleic acid,

wherein said first liquid flow channel comprises a second liquid flow channel connected to a third liquid flow channel wherein said second liquid flow channel comprises said leading electrolyte and said third liquid flow channel comprises said trailing electrolyte; and
a reservoir fluidly coupled to said first, second, or third liquid flow channels.

27. An isotachophoresis (ITP) apparatus comprising:
at least a first liquid flow channel containing:
a) a leading electrolyte,
b) a trailing electrolyte,
c) a catalyst, an enzyme, or a denaturant, and
d) a ribonucleic acid.

28. An isotachophoresis (ITP) apparatus comprising:
at least a first liquid flow channel containing:
i. a leading electrolyte;
ii. a trailing electrolyte; and
iii. a denaturant, wherein said denaturant comprises urea or formamide; and
iv. a nucleic acid.

29. An isotachophoresis (ITP) apparatus comprising at least a first liquid flow channel comprising:
(a) a first zone comprising a trailing electrolyte buffer comprising a trailing electrolyte;
(b) a second zone comprising a sample comprising an analyte, wherein said analyte comprises a ribonucleic acid;
(c) a third zone comprising a leading electrolyte buffer comprising a leading electrolyte that is faster than said trailing electrolyte; and
(d) a catalyst, enzyme, or denaturant,
wherein said analyte has an electrophoretic mobility between said trailing electrolyte and said leading electrolyte, and
wherein said catalyst, enzyme, or denaturant, is (i) positioned ahead of said analyte if said catalyst, enzyme, or denaturant, has an electrophoretic mobility slower than said electrophoretic mobility of said analyte; or (ii) positioned behind said analyte if said catalyst, enzyme, or denaturant, has an electrophoretic mobility faster than said electrophoretic mobility of said analyte.

30. The apparatus of claim 29 , wherein said leading electrolyte buffer comprises said catalyst or enzyme.

31. The apparatus of claim 29 , wherein said first liquid flow channel further comprises at least one primer, oligonucleotide, or dNTP.

32. The apparatus of claim 29 , wherein said catalyst, enzyme, or denaturant comprises a denaturant.

33. The apparatus of claim 29 , wherein said catalyst, enzyme, or denaturant comprises an enzyme.

34. The apparatus of claim 29 , wherein said catalyst, enzyme, or denaturant comprises a catalyst.

Assignees

• Univ Leland Stanford Junior

Inventors

• Santiago Juan G
• Persat Alexandre

Classifications

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12M1/42

  Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• G01N27/44791

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

• C12Q2565/125

  Electrophoretic separation · CPC title

• G01N27/44765 Primary

  of the counter-flow type · CPC title