Method for reducing the linear dimension necessary for high resolution electrophoretic separation

• US 6,013,166 A
• Filed: 04/28/1994
• Issued: 01/11/2000
• Est. Priority Date: 05/09/1991
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Heller, Michael J.
• Primary Examiner(s): Beisner, William H.
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/234,637
• Time in Patent Office: 2,084 Days
• Field of Search: 204/182.8, 204/299 R, 204/456, 204/458, 204/468, 204/467, 204/469, 204/607, 204/616, 204/618, 204/606
• US Class Current: 204/469
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44756 Apparatus specially adapted therefor

Abstract

The present invention relates to an electrophoretic method to effect differential net migration, the extent of said migration being dependent on molecular size, of electrically charged macromolecules through a gel support in a single dimension, which method comprises subjecting electrically charged macromolecules applied to a gel support to an electric field oriented along a single axis within the gel for a time period sufficient to effect migration in the direction of the oriented field and form a separation pattern in order of the respective molecular weights of the macromolecules in a distance of about 0.5 to about 20 millimeters, said gel support comprising about 3 to about 40 percent polyacrylamide, said electric field being applied in an amount of about 5 to about 100 volts per millimeter of gel support along the axis length, and said gel support having a width perpendicular to said axis of about 0.1 to 1.5 millimeters.

Claims

1. An electrophoretic method of effecting differential net migration and separation, in a time period less than 10 minutes over a distance of about 0.5 millimeters to about 2.0 centimeters in the direction of an electrical field oriented along a single linear axis within a gel support comprising a polyacrylamide containing about 20-30% acrylamide and about 5-11crosslinker and having a thickness perpendicular to said axis of about 0.1-1.5 millimeters, of electrically charged, differentially-sized linear macromolecules, the extent of said migration and separation of said macromolecules being dependent on their molecular sizes, which method comprises:

(1) applying said macromolecules to said gel support, and

(2) applying to said linear axis of said gel support containing said macromolecules an electrical field comprising about 5-100 volts per millimeter of gel in said gel support to effect migration and separation of said macromolecules into a pattern within said gel support, said macromolecules being ordered within said pattern by their respective sizes.

2. The method of claim 1 for effecting differential net migration and separation over a distance of about 2 to 15 millimeters.

3. The method of claim 1 for effecting differential net migration and separation in a device having a thickness perpendicular to said axis of about 0.25 to 1 millimeter.

4. The method of claim 1 wherein the crosslinker is N, N'"'"'-methylenebisacrylamide.

5. An electrophoretic method of effecting differential net migration and separation, in a time period less than 10 minutes over a distance of about 0.5 millimeters to about 2.0 centimeters in the direction of an electrical field oriented along a single linear axis within a gel support comprises a polyacrylamide containing about 30-40% acrylamide and about 8-12% cross-linker and having a thickness perpendicular to said axis of about 0.1-1.5 millimeters, of electrically charged, differentially-sized linear macromolecules, the extent of said migration and separation of said macromolecules being dependent on their molecular sizes, which method comprises:

(1) applying said macromolecules to said gel support, and

(2) applying to said linear axis of said gel support containing said macromolecules an electrical field comprising about 5-100 volts per millimeter of gel in said gel support to effect migration and separation of said macromolecules into a pattern within said gel support, said macromolecules being ordered within said pattern by their respective sizes.

6. The method of claim 5 for effecting differential net migration and separation over a distance of about 2 to 15 millimeters.

7. The method of claim 5 for effecting differential net migration and separation in a device having a thickness perpendicular to said axis of about 0.25 to 1 millimeter.

8. The method of claim 5 wherein the crosslinker is N, N'"'"'-methylenebisacrylamide.

9. An electrophoretic method for effecting the differential net migration and separations a distance less than about 2.5 centimeters, of nucleic acid fragments which range in size from 2 nucleotides to substantially 10,000 nucleotides using restrictive pore media with pore limit diameters between substantially 2.0 nanometers and 10 nanometers, the method comprising the steps of:

applying the macromolecules to the gel support, and

applying an electric field along the linear axis of said gel support for a time period and in an amount sufficient to effect the net migration and separation of the nucleic acid fragments in the direction of the electric field and within the gel support.

10. A method of claim 9 for electrophoretic separation of nucleic acid fragments which range in size from 2 nucleotides to substantially 500 nucleotides using restrictive pore media with pore limit diameters between substantially 2.0 nanometers and 5.0 nanometers.

11. A method of claim 9 for electrophoretic separation of nucleic acid fragments which range in size from substantially 10 nucleotides to substantially 2,000 nucleotides using restrictive pore media with pore limit diameters between 2.0 nanometers and substantially 6.5 nanometers.

12. An electrophoretic method for effecting the differential net migration and separation, in a distance less than about 2.5 centimeters, of nucleic acid fragments which range in size from substantially 50 nucleotides to at least 10,000 nucleotides using restrictive pore limit media with pore diameters between 4.2 nanometers and 10.0 nanometers, the method comprising the steps of:

applying the macromolecules to the gel support, and

applying an electric field along the linear axis of said gel support for a time period and in an amount sufficient to effect the net. migration and separation of the nucleic acid fragments in the direction of the electric field and within the gel support.

13. An electrophoretic method for effecting the differential net migration and separation, in a distance less than about 2.5 centimeters, of nucleic acid fragments which range in size from 2 nucleotides to substantially 1000 nucleotides using polyacrylamide gel media where the concentration is in the range from 40%T/12% C. to 20%T/5%C., the method comprising the steps of:

applying the macromolecules to the gel support, and

applying an electric field along the linear axis of said gel support for a time period and in an amount sufficient to effect the net migration and separation of the nucleic acid fragments electrically charged macromolecules in the direction of the electric field and within the gel support.

14. An electrophoretic method for effecting the differential net migration and separation, in a distance less than about 2.5 centimeters, of nucleic acid fragments which range in size from substantially 25 nucleotides to substantially 2,000 nucleotides using polyacrylamide gel media with a concentration in the range from 30%T/l1%C. to 15%T/14%C, the method comprising the steps of:

applying the macromolecules to the gel support, and

applying an electric field along the linear axis of said gel support for a time period and in an amount sufficient to effect the net migration and separation of the nucleic acid fragments in the direction of the electric field and within the gel support.

15. An electrophoretic method for effecting the differential net migration and separation in a distance less than about 2.5 centimeters, of nucleic acid fragments which range in size from substantially 200 nucleotides to at least 10,000 nucleotides using polyacrylamide gel media where the concentration is in the range from substantially 25%T/10%C to substantially 10%T/3%C, the method comprising the steps of:

applying the macromolecules to the gel support, and

applying an electric field along the linear axis of said gel support for a time period and in an amount sufficient to effect the net migration and separation of the nucleic acid fragments in the direction of the electric field and within the gel support.

16. An electrophoretic method for effecting the differential net migration and separation, in a time period less than substantially 10 minutes and in a distance less than about 2.5 centimeters, of linear macromolecules having a range of sizes less than substantially 10,000 bases, the linear macromolecules having net charges proportional to their molecular weight, said charged macromolecules exhibiting differential net migration within a gel support in a single linear dimension to an extent dependent on the molecular size of the linear macromolecules when subjected to an electric field, the gel support characterized by a pore size which is less than the radius of gyration of the linear macromolecules, the method comprising the steps of:

(1) applying the linear macromolecules to the gel support, and

(2) applying an electric field oriented along the single linear dimension of said gel support for substantially 10 minutes or less and in an amount sufficient to form a separation pattern along the single linear dimension comprised of a distribution of the linear macromolecules within the gel support ordered according to their respective molecular sizes.

17. The method of claim 16 wherein said time period is less than 2 minutes.

18. The method of claim 16 wherein said electric field comprises about 5-100 volts per millimeter of gel in said gel support.

19. The method of claim 16 wherein said time period is less than 10 minutes and said electric field comprises about 5-100 volts per millimeter of gel in said gel support.

20. The method of claim 16 or 19 wherein the gel support is formed of a total of about 12-40% of polyacrylamide with 4 to 12% of cross-linking agent to form a cross-linked polyacrylamide gel.

21. The method of claim 20 wherein the gel support is formed of a total of about 15-35% of polyacrylamide with 4 to 12% cross-linking agent to form a cross-linked polyacrylamide gel.

22. The method of claim 16 for the electrophoretic separation of macromolecules including nucleic acid, fragments which range in size from 2 nucleotides to substantially 10,000 nucleotides using restrictive pore media with pore diameters between 2.0 nanometers and 10.0 nanometers.

23. The method of claim 22 for the electrophoretic separation of nucleic acid fragments which range in size from 2 nucleotides to substantially 1,000 nucleotides using restrictive pore media with pore diameters between 2.0 nanometers and 5.0 nanometers.

24. The method of claim 22 for the electrophoretic separation of nucleic acid fragments which range in size from substantially 10 nucleotides to substantially 2,000 nucleotides using restrictive pore media with pore diameters between 2.0 nanometers and 6.7 nanometers.

25. The method of claim 16 wherein the gel is formed with a thickness of about 0.1 to 1.5 millimeters in a direction perpendicular to the linear dimension.

26. The method of claim 25 wherein gel is formed to be about 0.25 to 1.0 millimeters thick in a direction perpendicular to the linear dimension.

27. The method of claim 25 wherein the gel is formed to be cylindrical.

28. The method of claim 16 further comprising the step of detecting the relative location of each separated macromolecule species within the sample.

29. The method of claim 28 wherein the detecting is performed with an associated integrated imaging system.

30. The method of claim 28 wherein the detecting is performed with an associated electronic imaging system.

31. The method of claim 30 wherein a microchannel plate detector performs the electronic imaging.

32. The method of claim 30 wherein a charge coupled device performs the electronic imaging.

33. The method of claim 30 wherein a intensified charge coupled device performs the electronic imaging.

34. The method of claim 30 wherein a cooled charge coupled device performs the electronic imaging.

35. The method of claim 30 wherein a photon counting device performs the electronic imaging.

36. The method of claim 30 wherein a silicon intensified tube performs the electronic imaging.

37. The method of claims 16 or 28 further comprising the step of isolating at least one species of macromolecules from the sample.

38. The method of claim 16 wherein the gel is formed within a capillary tube.

39. The method of claim 16 wherein the gel is formed in a microslab arrangement.

40. The method of claim 16 the gel is formed in a microchannel arrangement.

41. The method of claim 40 wherein the microchannel is formed with a width less than 0.1 mm.

42. The method of claim 40 wherein the microchannel is formed in glass.

43. The method of claim 40 wherein the microchannel arrangement comprises multiple channels.

44. The method of claim 43 wherein microchannels are formed with widths less than 0.1 mm.

45. The method of claim 44 wherein microchannels are formed in glass.

46. The method of claim 16 wherein the actual gel length along the linear dimension is formed to be from about 1 to 25 millimeters.

47. The method of claim 46 wherein the actual gel length along the linear dimension is from about 2 to 15 millimeters.

48. The method of claim 46 wherein the actual gel length along the linear dimension is from about 2 to 10 millimeters.

49. The method of claims 16 or 19 wherein the gel support is agarose.

50. The method of claims 16 or 19 wherein the gel support comprises starch.

51. The method of claims 16 or 19 wherein the gel support comprises a hydrogel.

52. The method of claim 28 wherein said detectable separation is achieved within a 40×

or less magnification field.

53. The method of claim 16 wherein electric field is switched on and off.

54. The method of claim 16 wherein the gel is selected from the group consisting of:

starch, natural hydrogels and synthetic hydrogels.