Highly derivatized agarose conformational nucleic acid separation

• US 5,641,626 A
• Filed: 10/29/1993
• Issued: 06/24/1997
• Est. Priority Date: 10/29/1993
• Status: Expired due to Term

• Current Assignee: Cambrex Bio Science Rockland, Inc.
• Original Assignee: FMC Corporation
• Inventors: White, Hugh, Dumais, Maxine, Kusukawa, Noriko
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/146,286
• Time in Patent Office: 1,334 Days
• Field of Search: 435/6, 252/315.3, 436/94
• US Class Current: 516/105
• CPC Class Codes: C08L 5/12 Agar or agar-agar , i.e. mixture of agarose and agaropectin; Derivatives thereof
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Compositions for the gel electrophoresis separation of conformational isomers of nucleic acids and agarose compositions useful in such compositions. The compositions use compositions comprising: at least one aqueous gel which is at least one highly derivatized agarose derivatized sufficiently to reduce its gelling temperature (Tg) to below 17° C., present in 40 wt % or more based upon the gel total weight, in admixture with at least one high gel-strength polysaccharide aqueous gel other than polyacrylamide in a balance to 100% total gel weight; and an electrophoretic buffer, present in an electrophoretic buffer--effective amount. The invention compositions comprise at least one aqueous gel comprising at least one agarose derivatized sufficiently to reduce its gelling temperature (Tg) to below 9° C., present in 40 wt % or more based upon the gel total weight, in admixture with at least one high strength non-acrylamide polysaccharide present in a balance to 100% total gel weight; and an electrophoretic buffer, present in an electrophoretic buffer--effective amount.

Claims

1. A composition for the electrophoretic separation of conformational nucleic acid isomers comprising:

A) an aqueous gel comprising

(1) at least one agarose derivatized to the extent that it has become non-gelling or sufficiently to reduce its gelling temperature (Tg) as measured at 0.8 wt % concentration to below 9°

C., present in 40 wt % or more based upon the gel total weight, in admixture with

(2) at least one high strength agarose matrix gel present in an amount which together with the derivatized agarose gives a 100 wt % total gel weight; and

B) an electrophoretic buffer, present in an electrophoretic buffer--effective amount.

3. The composition of claim 1 or 2 wherein the highly derivatized agarose is substituted with a:

C_1-4 alkyl;

hydroxy, dihydroxy or polyhydroxy-(C_1-5) alkyl;

an ester or ether of the agarose with a C_1-4 neutral moiety having 0 or 1 to 3 oxygen atoms;

or is a mixture of these derivatized agaroses.

4. The composition of claim 3 wherein the highly derivatized agarose is hydroxyethyl agarose.

5. The composition of claim 3 wherein the highly derivatized agarose is present in up to 90 wt %.

6. The composition of claim 3 wherein the highly derivatized agarose is present in 60 to 90 wt %.

7. The composition of claim 3 wherein the highly derivatized agarose is present in 70 to 90 wt %.

8. The composition of claim 3 wherein the highly derivatized agarose is present in about 80 wt %.

9. The composition of claim 3 wherein the derivatized agarose has a gelling temperature of from the lowest temperature at which gellation will occur to less than 9°

C.

10. The composition of claim 3 wherein the derivatized agarose has a gelling temperature of 0°

C. to less than 9°

C.

11. The composition of claim 3 wherein the derivatized agarose has a gelling temperature of 2°

C. to less than 9°

C.

12. The composition of claim 3 wherein the derivatized agarose has a gelling temperature of 6°

C. to less than 9°

C.

13. The composition of claim 3 wherein the derivatized agarose has a gelling temperature of 6°

C. to 8°

C.

14. The composition of claim 11 or 12 wherein:

A) said aqueous gel consists essentially of;

(1) hydroxyethyl agarose having a gelling temperature of from 6°

C. to less than 9°

C. and present in about 70 wt % to about 90 wt %; and

(2) a high gel-strength hydrocolloid matrix gel which is an agarose present in an amount which together with the hydroethyl agarose gives a 100 wt % total gel weight.

15. The composition of claim 1 or 2 wherein the high gel-strength agarose matrix gel has a gel strength of at least 500 g/cm at 1% w/w concentration.

16. The composition of claim 1 or 2 wherein the total gel concentration in water of the agaroses is from 2% to 10%.

27. The method of claim 5 wherein the derivatized agarose has a gelling temperature below 17°

C.

32. The method of claim 1 wherein the high gel-strength agarose matrix gel has a gel strength of at least 500 g/cm at 1% w/w concentration.

33. The method of claim 1 wherein the total gel concentration in water of the agaroses is from 2% to 10%.

2. A composition for the electrophoretic detection of point mutations by single strand conformational polymorphism (SSCP) analysis or heteroduplex/homoduplex analysis, or specific DNA-protein interactions by gel shift analysis comprising:

A) an aqueous gel comprising

(1) at least one agarose derivatized sufficiently to reduce its gelling temperature (Tg) as measured at 0.8 wt % concentration to below 9°

C., present in 40 wt % or more based upon the gel total weight, in admixture with

(2) at least one high strength agarose matrix gel present in an amount which together with the derivatized agarose gives a 100 wt % total gel weight; and

B) an electrophoretic buffer, present in an electrophoretic buffer--effective amount.

17. A method for the electrophoretic separation of conformational nucleic acid isomers employing an electrophoretic composition comprising:

A) an aqueous gel which is

(1) at least one highly derivitized agarose derivitized to the extent that it has become non-gelling or sufficiently to reduce its gelling temperature (Tg) as measured at 0.8 wt % concentration, in water to below 17°

C., present in 40 wt % or more based upon the gel total weight, in admixture with

(2) at least one high gel-strength agarose matrix gel in an amount which together with the derivitized agarose gives a 100 wt % total gel weight; and

B) an electrophoretic buffer, present in an electrophoretic buffer--effective amount.

18. A method for the detection of point mutations comprising Single Strand Conformational Polymorphism (SSCP) analysis by an assay using the method of claim 17.

19. A method for the detection of point mutations comprising Heteroduplex/Homoduplex analysis by an assay using the method of claim 17.

20. A method for the detection of specific DNA-protein interactions comprising Gel Shift analysis by an assay using the method of claim 17.

21. The method of claim 17, 18, 19, or 20, wherein the highly derivatized agarose is substituted with a:

C_1-4 alkyl;

hydroxy, dihydroxy or polyhydroxy-(C_1-5) alkyl;

an ester or ether of the agarose with a C_1-4 neutral moiety having 0 or 1 to 3 oxygen atoms;

or is a mixture of these derivatized agaroses.

22. The method of claim 21 wherein the highly derivatized agarose is hydroxyethyl agarose.

23. The method of claim 21 wherein the highly derivatized agarose is present in up to 90 wt %.

24. The method of claim 21 wherein the highly derivatized agarose is present in 60 to 90 wt %.

25. The method of claim 21 wherein the highly derivatized agarose is present in 70 to 90 wt %.

26. The method of claim 21 wherein the highly derivatized agarose is present in about 80 wt %.

28. The method of claim 21 wherein the derivatized agarose has a gelling temperature of 0°

C. to less than 17°

C.

29. The method of claim 21 wherein the derivatized agarose has a gelling temperature of 2°

C. to less than 17°

C.

30. The method of claim 21 wherein the derivatized agarose has a gelling temperature of 6°

C. to less than 9°

C.

31. The method of claim 23 wherein the derivatized agarose has a gelling temperature of 6°

C. to 8°

C.