Electrophoresis gels of enhanced selectivity

• US 5,840,877 A
• Filed: 09/05/1996
• Issued: 11/24/1998
• Est. Priority Date: 09/05/1996
• Status: Expired due to Term

• Current Assignee: Elchrom Scientific, Elechrom Scientific, AG
• Original Assignee: Guest Elchrom Scientific Co.
• Inventors: Kozulic, Branko
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/708,492
• Time in Patent Office: 810 Days
• Field of Search: 536/123.1, 536/124, 536/25.4, 526/304, 356/344, 204/456
• US Class Current: 536/25.4
• CPC Class Codes: C07K 1/26 Electrophoresis
• CPC Class Codes: C08F 220/56 Acrylamide; Methacrylamide
• CPC Class Codes: C08F 251/00 Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
• CPC Class Codes: C08F 265/00 Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture

Abstract

Electrophoresis gels of enhanced selectivity are produced by adding a preformed polymer to a polymerization solution containing at least one monomer and at least one cross-linker. The polymer must be of such a chemical composition and molecular weight that its presence during free-radical polymerization results in a new topology of gel polymers, so that under the influence of an electric field, such as during electrophoresis, the passage of long DNA molecules through the gel is retarded proportionally more than the passage of the short ones. The polymer needs to be added at specific ratio to the monomer and the cross-linker, and the enhanced selectivity is achievable only within a certain range of ratios of polymer(s), monomer(s) and cross linker(s). That range is dependent on all three essential gel components, the polymer, the monomer and the cross-linker. Some gels possessing enhanced selectivity were able to resolve one base pair differences between DNA fragments in the 100 bp range with the gel length of less than 10 cm.

Claims

1. A gel suited for use in electrophoretic separation of high molecular weight molecules comprising the free radical polymerization reaction product of a mixture of:

at least one monomer; and

at least one vinyl cross linker reactive with said monomer and a polymer of said monomer sufficient to cross link said polymer; and

at least one preformed polymer;

wherein the concentration of a combination of said monomer and said cross linker is at least about 4% (w/v), and the concentration of said preformed polymer is about 0.005 to 2% (w/v);

wherein said gel selectively retards the electrophoretic migration rate of larger macro molecules, as compared to the migration rate of smaller macro molecules, from among DNA fragments of about 100 bp to 3,000 bp, by at least 5 fold as compared to the migration rates of the same molecules under the same conditions in a gel of substantially the same concentration and comprising the free radical polymer reaction product of a solution containing identical concentrations of said monomer and said cross linker but in the absence of said preformed polymer,

provided that when the monomer is acrylamide, and the cross linker is N,N'"'"'-methylene-bis-acrylamide, the preformed polymer is not agarose.

2. A gel of claim 1 comprising one monomer.

3. A gel of claim 2, wherein the monomer is N-acryloyl-tris(hydroxymethyl)aminomethane.

4. A gel of claim 2, wherein the monomer is acrylamide.

5. A gel of claim 2, wherein the monomer is N-acryloyl-1-amino-1-deoxy-D-galactitol.

6. A gel of claim 1, containing more than one monomer.

7. A gel of claim 6, wherein the monomers are acrylamide and N-acryloyl tris(hydroxymethyl)aminomethane.

8. A gel of claim 1 containing one cross-linker.

9. A gel of claim 8, wherein the cross-linker is N,N'"'"'-methylene-bis-acrylamide.

10. A gel of claim 8, wherein the cross-linker is dihydroxyethylene-bis-acrylamide.

11. A gel of claim 8, wherein the cross-linker is piperazine-di-acrylamide.

12. A gel of claim 1 containing more than one cross-linker.

13. A gel of claim 12, wherein the cross-linkers are N,N'"'"'-methylene-bis-acrylamide and dihydroxyethylene-bis-acrylamide.

14. A gel of claim 1 containing one polymer.

15. A gel of claim 14, wherein the polymer is a polysaccharide.

16. A gel of claim 15, wherein the polymer is hydroxyethyl cellulose.

17. A gel of claim 15, wherein the polymer is hydroxypropylmethyl cellulose.

18. A gel of claim 15, wherein the polymer is methyl cellulose.

19. A gel of claim 15, wherein the polymer is agarose.

20. A gel of claim 15, wherein the polymer is hydroxyethyl agarose.

21. A gel of claim 15, wherein the polymer is galactomannan.

22. A gel of claim 15, wherein the polymer is dextran.

23. A gel as claimed in claim 15 wherein said preformed polymer has a polydispersity, and wherein said polymer has been subjected to fractionation under conditions sufficient to reduce the polydispersity thereof.

24. A gel of claim 23, wherein the fractionation method is gel filtration.

25. A gel of claim 23, wherein the polymer is hydroxyethyl cellulose.

26. A gel of claim 1 containing more than one polymer.

27. A gel of claim 26, wherein the polymers are hydroxyethyl cellulose and hydroxyethyl agarose.

28. A gel of claim 1 containing a synthetic polymer.

29. A gel of claim 28, wherein the polymer is polyacrylamide.

30. A gel of claim 28, wherein the polymer is poly-N-acryloyl-tris(hydroxymethyl)-aminomethane.

31. A method for preparing a gel suited for use in electrophoretic separation of high molecular weight molecules comprising the steps of:

dissolving so much of at least one monomer, at least one vinyl cross linker, and at least one preformed polymer in a mutual solvent such that the concentration of said preformed polymer is about 0.005 to 2% (w/v) and the concentration of a combination of said monomer plus said cross linker is at least about 4% (w/v), wherein said monomer and cross linker contain vinyl groups, provided that when said monomer is acrylamide, and said cross linker is N,N'"'"'-methylene-bis-acrylamide, said polymer is not agarose; and

free radical polymerizing and cross linking said monomer and said cross linker in said solution through said vinyl groups sufficient to form a gel;

wherein said polymerization is carried out under conditions sufficient to form a gel which selectively retards the electrophoretic migration rate of larger macro molecules, as compared to the migration rate of smaller macro molecules, from among DNA fragments of about 100 bp to 3,000 bp, by at least 5 fold as compared to the migration rates of the same molecules under the same conditions in a gel of substantially the same concentration and composition comprising the free radical polymerization reaction product of a solution containing identical concentrations of said monomer and said cross linker but in the absence of said preformed polymer.

32. A method of claim 31, wherein the gel contains one monomer.

33. A method of claim 32, wherein the monomer is N-acryloyl-tris(hydroxymethyl)aminomethane.

34. A method of claim 32, wherein the monomer is acrylamide.

35. A method of claim 32, wherein the monomer is N-acryloyl-1-amino-1-deoxy-D-galactitol.

36. A method of claim 31, wherein the gel contains more than one monomer.

37. A method of claim 36, wherein the monomers are acrylamide and N-acryloyl-tris(hydroxymethyl)aminomethane.

38. A method of claim 31, wherein the gel contains one cross-linker.

39. A method of claim 38, wherein the cross-linker is N,N'"'"'-methylene-bis-acrylamide.

40. A method of claim 38, wherein the cross-linker is dihydroxyethylene-bis-acrylamide.

41. A method of claim 38, wherein the cross-linker is piperazine-di-acrylamide.

42. A method of claim 31, wherein the gel contains more than one cross-linker.

43. A method of claim 42, wherein the cross-linkers are N,N'"'"'-methylene-bis-acrylamide and dihydroxyethylene-bis-acrylamide.

44. A method of claim 31, wherein the gel contains one polymer.

45. A method of claim 44, wherein the polymer is a polysaccharide.

46. A method of claim 45, wherein the polymer is hydroxyethyl cellulose.

47. A method of claim 45, wherein the polymer is hydroxypropylmethyl cellulose.

48. A method of claim 45, wherein the polymer is methyl cellulose.

49. A method of claim 45, wherein the polymer is agarose.

50. A method of claim 45, wherein the polymer is hydroxyethyl agarose.

51. A method of claim 45, wherein the polymer is galactomannan.

52. A method of claim 45, wherein the polymer is dextran.

53. A method as claimed in claim 45 further including the step of subjecting said preformed polymer to fractionation, prior to combining said preformed polymer with said monomer and said cross linker, under conditions which are sufficient to reduce the polydispersity of said polymer.

54. A method of claim 53, wherein the fractionation method is gel filtration.

55. A method of claim 53, wherein the polymer is hydroxyethyl cellulose.

56. A method of claim 31, wherein the gel contains more than one polymer.

57. A method of claim 56, wherein the polymers are hydroxyethyl cellulose and hydroxyethyl agarose.

58. A method of claim 31, wherein the gel contains a synthetic polymer.

59. A method of claim 58, wherein the polymer is polyacrylamide.

60. A method of claim 58, wherein the polymer is poly-N-acryloyl-tris(hydroxymethyl)aminomethane.

61. A method of electrophoretic separation of large molecules of about the size and charge of DNA molecules which comprises:

forming a gel comprising the polymerization reaction product of a mixture of;

at least one monomer; and

at least one cross linker reactive with said monomer and a polymer of said monomer sufficient to cross link said polymer; and

at least one preformed polymer;

wherein the concentration of a combination of said monomer and said cross linker is at least about 4% (w/v), and the concentration of said preformed polymer is about 0.005 to 2% (w/v);

forming said gel into electrophoretic passageway means;

disposing a mixture of said molecules at one end of said passageway means; and

subjecting said mixture of molecules to an electrophoretic current and voltage for a time and at a temperature sufficient to cause said molecules to migrate through said gel a distance which is related to molecular weights of said molecules;

wherein said gel selectively retards electrophoretic migration rates of larger macro molecules, as compared to migration rates of smaller macro molecules, from among molecules of about 100 bp to 3,000 bp, by at least 5 fold as compared to the migration rates of the same molecules under the same conditions in a gel of substantially the same concentration and composition comprising the free radical polymerization reaction product of said monomer and said cross linker but in the absence of said preformed polymer.

62. An electrophoretic method of claim 61, wherein the gel contains one monomer.

63. An electrophoretic method of claim 62, wherein the monomer is N-acryloyl-tris(hydroxymethyl)aminomethane.

64. An electrophoretic method of claim 62, wherein the monomer is acrylamide.

65. An electrophoretic method gel of claim 62, wherein the monomer is N-acryloyl-1-amino-1-deoxy-D-galactitol.

66. An electrophoretic method of claim 61, wherein the gel contains more than one monomer.

67. An electrophoretic method of claim 66, wherein the monomers are acrylamide and N-acryloyl-tris(hydroxymethyl)aminomethane.

68. An electrophoretic method of claim 61, wherein the gel contains one cross-linker.

69. An electrophoretic method of claim 68, wherein the cross-linker is N,N'"'"'-methylene-bisacrylamide.

70. An electrophoretic method of claim 68, wherein the cross-linker is dihydroxyethylene-bisacrylamide.

71. An electrophoretic method of claim 68, wherein the cross-linker is piperazine-diacrylamide.

72. An electrophoretic method of claim 61, wherein the gel contains more than one cross linker.

73. An electrophoretic method of claim 72, wherein the cross-linkers are N,N'"'"'-methylene-bisacrylamide and dihydroxyethylene-bis-acrylamide.

74. An electrophoretic method of claim 61, wherein the gel contains one polymer.

75. An electrophoretic method of claim 74, wherein the polymer is a polysaccharide.

76. An electrophoretic method of claim 75, wherein the polymer is hydroxyethyl cellulose.

77. An electrophoretic method of claim 75, wherein the polymer is hydroxypropylmethyl cellulose.

78. An electrophoretic method of claim 75, wherein the polymer is methyl cellulose.

79. An electrophoretic method of claim 75, wherein the polymer is agarose.

80. An electrophoretic method of claim 75, wherein the polymer is hydroxyethyl agarose.

81. An electrophoretic method of claim 75, wherein the polymer is galactomannan.

82. An electrophoretic method of claim 75, wherein the polymer is dextran.

83. The electrophoretic method as claimed in claim 61 wherein said preformed polymer has been subjected to fractionation under conditions sufficient to reduce the polydispersity of said polymer before it is admixed with said monomer and said cross linker.

84. An electrophoretic method of claim 83, wherein the fractionation method is gel filtration.

85. An electrophoretic method of claim 83, wherein the polymer is hydroxyethyl cellulose.

86. An electrophoretic method of claim 61, wherein the gel contains more than one polymer.

87. An electrophoretic method of claim 86, wherein the polymers are hydroxyethyl cellulose and hydroxyethyl agarose.

88. An electrophoretic method of claim 61, wherein the gel contains a synthetic polymer.

89. An electrophoretic method of claim 88, wherein the polymer is polyacrylamide.

90. An electrophoretic method of claim 88, wherein the polymer is poly-N-acryloyl-tris(hydroxymethyl)aminomethane.