Agarose compositions for nucleic acid sequencing

• US 5,455,344 A
• Filed: 09/03/1993
• Issued: 10/03/1995
• Est. Priority Date: 09/03/1993
• Status: Expired due to Term

• Current Assignee: Cambrex Bio Science Rockland, Inc.
• Original Assignee: FMC Corporation
• Inventors: Harper, David L., Ostrovsky, Mikhail V., Morgan, Jonathan H., Renn, Donald W., Nochumson, Samuel, Snow, William C.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Kunz, Gary L.
• Application Number:
  US08/116,662
• Time in Patent Office: 760 Days
• Field of Search: 536/1.1, 536/25.4, 536/123.1, 935/77
• US Class Current: 536/123.1
• CPC Class Codes: C08L 5/00 Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture

Abstract

A nucleic acid sequencing gel comprising 2 to 10% of a gelling polysaccharide, preferably agarose; a denaturing agent, a non-gelling additive, preferably glycerol, and electrophoretic buffers. This composition is capable of resolving nucleic acid fragments having at least 50 bases and which differ from each other by at least one base.

Claims

1. An aqueous nucleic acid sequencing gel consisting essentially of:

[a] 2 to 10 wt % of one or more gelling polysaccharides selected from the group consisting of agarose, deacetylated konjac glucomannan, and hydroxyethyl curdlan;

[b] 2 to 95 wt % of one or more denaturing agents;

[c] 0 to 50 wt % of one or more non-gelling additives;

[d] 0.2 to 6 wt % of one or more electrophoretic buffers; and

[e] water sufficient to 100 wt %;

wherein the gel is capable of resolving nucleic acid fragments having at least 50 bases which differ from each other by at least one base.

3. The composition of claim 1 wherein the gelling polysaccharide is agarose.

4. The composition of claim 1 wherein the gelling polysaccharide is deacetylated konjac glucomannan.

5. The composition of claim 1 wherein the gelling polysaccharide is hydroxyethyl curdlan.

6. The composition of claim 1 wherein the gelling polysaccharide is agarose extracted from the Pterocladia species of seaweed.

7. The composition of claim 1 wherein the denaturing agent is:

urea;

biuret;

C_1-6 alkyl urea;

dimethylsulfoxide;

C_1-6 monoaldehyde;

C_1-6 dialdehyde;

formamide;

C_1-6 alkyl formamide;

dimethylsulfoxide;

pyrrolidinone;

C_1-6 alkyl pyrrolidinone;

C_2-4 glycol;

or a mixture thereof.

8. The composition of claim 1 wherein the denaturing agent is:

urea;

formamide;

dimethylformamide;

dimethylsulfoxide;

or a mixture thereof.

9. The composition of claim 1 wherein the denaturing agent is:

urea;

formamide;

dimethylformamide;

or a mixture thereof.

10. The composition of claim 1 wherein the denaturing agent is urea.

11. The composition of claim 1 wherein the non-gelling additive is present and is:

glycerol;

C_1-4 glycol;

C_1-4 polyglycol;

dextran;

locust bean gum;

depolymerized agarose;

or a mixture thereof.

12. The composition of claim 1 wherein the non-gelling additive is present and is:

glycerol, ethylene glycol, erythritol, locust bean gum;

or a mixture thereof.

13. The composition of claim 1 wherein the non-gelling additive is present and is:

glycerol;

ethylene glycol;

or a mixture thereof.

14. The composition of claim 1 wherein the electrophoretic buffer is a biological buffer and is tris-R-EDTA where R is:

borate;

phosphate;

taurine;

acetate;

carbonate;

or glycine;

or a mixture of such buffers.

15. The composition of claim 1 wherein the electrophoretic buffer is a biological buffer and is tris-R-EDTA where R is:

borate;

phosphate;

taurine;

or acetate;

or a mixture of such buffers.

16. The composition of claim 1 wherein the electrophoretic buffer is tris-borate-EDTA.

17. The composition of claim 1 wherein:

(i) the gelling polysaccharide is agarose, konjac glucomannan, hydroxyethyl curdlan, or a mixture thereof;

(ii) the denaturing agent is;

urea;

biuret;

C_1-6 alkyl urea;

dimethylsulfoxide;

C_1-6 monoaldehyde;

C_1-6 dialdehyde;

formamide;

C_1-6 alkyl formamide;

pyrrolidinone;

C_1-6 alkyl pyrrolidinone;

C_2-4 glycol;

or a mixture thereof; and

(iii) the electrophoretic buffer is a biological buffer and is tris-R-EDTA

where R is;

borate;

phosphate;

taurine;

acetate;

carbonate;

or

glycine;

or a mixture of such buffers.

18. The composition of claim 17 wherein:

(iv) the non-gelling additive is present and is;

glycerol;

C_1-4 glycol;

C_1-4 polyglycol;

dextran;

locust bean gum;

depolymerized agarose;

or a mixture thereof.

19. The composition of claim 1 wherein:

(i) the gelling polysaccharide is agarose, hydroxyethyl curdlan, deacetylated konjac glucomannan, or a mixture thereof;

(ii) the denaturing agent is;

urea;

formamide;

dimethylformamide;

dimethylsulfoxide;

or a mixture thereof; and

(iii) the electrophoretic buffer is a biological buffer and is tris-R-EDTA where R is;

borate;

phosphate;

taurine;

or acetate;

or a mixture of such buffers.

20. The composition of claim 19 wherein:

(iv) the non-gelling additive is present and is;

glycerol, ethylene glycol, erythritol, locust bean gum;

or a mixture thereof.

21. The composition of claim 1 wherein:

(i) the gelling polysaccharide is agarose extracted from the Pterocladia species of seaweed;

(ii) the denaturing agent is urea; and

the electrophoretic buffer is tris-borate-EDTA.

22. The composition of claim 21 wherein:

(iv) the non-gelling additive is present and is;

glycerol;

ethylene glycol;

or a mixture thereof.

23. The composition of claim 1 wherein the ingredients are present in the amounts:

(a) gelling polysaccharide;

2 to 8 wt %;

(b) denaturing agent;

10 to 70 wt %; and

(d) electrophoretic buffer;

0.3 to 5 wt %.

24. The composition of claim 23 wherein the non-gelling additive is present in the amount 2 to 30 wt %.

25. The composition of claim 1 wherein the ingredients are present in the amounts:

(a) gelling polysaccharide;

3 to 6 wt %;

(b) denaturing agent;

12 to 60 wt %; and

(d) electrophoretic buffer;

0.4 to 4 wt %.

26. The composition of claim 23 wherein the non-gelling additive is present in the amount 5 to 20 wt %.

2. A dry combination of nucleic acid sequencing aqueous gel precursor ingredients consisting essentially of:

[a] 2 to 10 wt % of one or more gelling polysaccharides selected from the group consisting of agarose, deacetylated konjac glucomannan, hydroxylethyl curdlan;

[b] 2 to 95 wt % of one or more denaturing agents;

[c] 0 to 50 wt % of one or more non-gelling additives; and

[d] 0.2 to 6 wt % of one or more electrophoretic buffers;

wherein the gel resulting from the addition of water to 100 wt % to said combination is capable of resolving nucleic acid fragments having at least 50 bases which differ from each other by at least one base.