Methods and compositions for performing molecular separations

• US 20020166768A1
• Filed: 05/24/2002
• Published: 11/14/2002
• Est. Priority Date: 12/17/1997
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Dubrow, Robert S.
• Granted Patent: US 7,081,190 B2
• Time in Patent Office: Days
• US Class Current: 204/454
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44752 Controlling the zeta potential, e.g. by wall coatings
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

The present invention provides methods of electrophoretically separating macromolecular species, as well as compositions and systems useful in carrying out such methods. Specifically, the methods of the present invention comprise providing a substrate that has at least a first capillary channel disposed therein. The surface of the channel has a first surface charge associated therewith, and is filled with a water soluble surface adsorbing polymer solution that bears a net charge that is the same as the charge on the capillary surface.

Claims

1. A method of separating macromolecules by capillary electrophoresis, comprising:

providing a substrate comprising at least a first capillary channel disposed therein, a surface of the channel having a first surface charge associated therewith;

filling said capillary channel with a water soluble hydrophilic polymer having a charge in solution that is the same as the first surface charge;

introducing a sample containing the macromolecules into one end of the capillary channel and;

applying a voltage gradient across the length of the capillary channel, whereby the macromolecules in the sample are separated in the capillary channel.

2. The method of claim 1, wherein the substrate has at least two intersecting microchannels.

3. The method of claim 2 wherein the polymer has a net charge of between about 0.01% and about 1%.

4. The method of claim 1, wherein the substrate provided in the providing step is a silica-based substrate.

5. The method of claim 4, wherein the substrate provided in the providing step comprises a silica substrate, and the polymer in the filling step comprises polydimethylacrylamide-co-acrylic acid.

6. The method of claim 1, wherein the substrate provided in the providing step comprises a solid polymeric substrate.

7. The method of claim 6, wherein the solid polymeric substrate is selected from the group of polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate, polytetrafluoroethylene.

8. The method of claim 1, wherein the sample contains a plurality of different nucleic acid sequences.

9. The method of claim 8, wherein the different nucleic acids comprise a plurality of different fragments of a target nucleic acid sequence.

10. The method of claim 9, wherein the different nucleic acids comprise a nested set of fragments of a target nucleic acid sequence.

11. The method of claim 10, wherein the each fragment in the nested set of fragments differs from at least one other fragment in the nested set by the addition or omission of a single nucleotide at a terminus of the fragment.

12. The method of claim 1, wherein the capillary channel provided in the providing step intersects and is fluidly connected with at least a second capillary channel disposed in the substrate.

13. The method of claim 1, wherein the capillary channel provided in the providing step intersects and is fluidly connected with at least second and third capillary channels disposed in the substrate.

14. The method of claim 13, wherein the applying step comprises simultaneously applying a voltage gradient across each of the first and second capillary channels, to transport the sample from the second channel into the first channel and to separate macromolecules in the sample in the first channel.

15. The method of claim 13, wherein the applying step comprises simultaneously applying a voltage gradient across each of the first, second and third capillary channels.

16. The method of claim 1, wherein the polymer solution comprises a polymer concentration of between about 0.01% and about 20% (w/v).

17. The method of claim 1, wherein the polymer solution has a viscosity of between about 2 centipoise and about 1000 centipoise.

18. The method of claim 1, wherein the polymer comprises a molecular weight from about 1 Kd, to about 5,000 Kd.

19. The method of claim 1, wherein the polymer is a polydimethylacrylamide polymer and the charged monomer is acrylic acid.

20. A method of separating macromolecules by capillary electrophoresis, comprising:

providing a substrate having a capillary channel disposed therein, a surface of the channel having a negative surface charge associated therewith;

filling said capillary channel with a water soluble hydrophilic polymer having a net charge, the charge being the same as the surface charge;

introducing a sample containing the macromolecules into one end of the capillary channel; and

applying a voltage gradient across the length of the capillary channel, whereby the macromolecules in the sample are separated in the capillary channel.

21. The method of claim 20, wherein the substrate has at least two intersecting capillary channels.

22. The method of claim 21, wherein the polymer has a net charge of between about 0.01% and about 1%.

25. The method of claim 24, wherein the substrate has at least two intersecting capillary channels disposed therein.

26. The method of claim 25, wherein the polymer has a net charge of between about 0.01% and about 1%.

27. The system of claim 24, wherein the net surface charge associated with the interior surfaces of the capillary channel is negative.

28. The system of claim 27, wherein the substrate is a silica substrate.

29. The system of claim 28, wherein the substrate is selected from a silica capillary tube and an etched planar silica substrate.

30. The system of claim 24, wherein the substrate comprises a solid polymeric substrate.

31. The system of claim 30, wherein the solid polymeric substrate is selected from the group of polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate, polytetrafluoroethylene.

32. The system of claim 24, wherein the substrate further comprises at least a second walled capillary channel disposed in the substrate, the second walled capillary channel intersecting and in fluid communication with the first walled capillary channel.

33. The system of claim 32, wherein the power source is electrically coupled to each of the fist and second capillary channels, the power supply simultaneously applying a voltage gradient across a length of each of the first and second capillary channels.

34. The system of claim 24, wherein the polymer solution comprises a polymer concentration in a range of from about 0.1% to about 10% (w/v).

35. The system of claim 24, wherein the polymer solution comprises a viscosity of between about 2 centipoise and about 1000 centipoise.

36. The system of claim 24, wherein the polymer is an acrylic polymer and the charged monomer subunits are selected from acrylic acid, bisacrylamidoacetic acid, 4,4-Bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid, 2-carboxyethylacrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid, 4-styrenesulfonic acid, 2-sulfoethyl methacrylate, 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt, 3-sulfopropyl methacrylate, vinylsulfonic acid, Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.

37. The system of claim 24, wherein the polymer comprises polydimethylacrylamide-co-acrylic acid.

38. The system of claim 24, wherein the polymer has a net negative charge.

39. The system of claim 24, wherein the polymer is made by the process of polymerizing dimethylacrylamide monomers in the presence acrylic acid, the acrylic acid being present at a concentration of between about 0.01 and 2% of a total monomer concentration.

40. The system of claim 24, wherein the first net surface charge is capable of supporting an electroosmotic mobility of a buffer comprising from about 1 mM to about 10 mM sodium borate buffer, at a pH of from about 7 to about 9, disposed in the walled capillary channel, the electroosmotic mobility being at least about 1×

10−

5 cm2V−

1s−

1.

23. A method of preparing a capillary channel for use in separating macromolecules, comprising:

filling the capillary channel with a polymer solution, wherein the polymer has a net charge that is the same as a net charge associated with interior surfaces of the capillary channel.

24. A system for separating macromolecules by capillary electrophoresis, comprising:

a substrate having at least a first capillary channel disposed therein, the channel having a net surface charge associated with interior surfaces of the channel;

a polymer disposed in the capillary channel, the polymer having a net charge, the net charge being the same as the net surface charge.

41. A system for separating nucleic acids by molecular weight, comprising:

a substrate having a capillary channel disposed therein, the channel having a charge associated with interior surfaces of the channel; and

a polymer disposed in the capillary channel, the polymer having a net charge, the net charge being the same as the net surface charge.

42. The system of claim 41, wherein the substrate has at least two intersecting microchannels therein.

43. The system of claim 42, wherein the polymer has a net charge of between about 0.01% and about 1%

44. A capillary for separating macromolecules by capillary electrophoresis, comprising:

a capillary channel disposed in a substrate, interior surface of the capillary having a first net surface charge associated therewith; and

a polymer disposed in the capillary channel, the polymer having a net charge, the net charge being the same as the first net surface charge.