Matrix modification in the electrophoretic separation of nucleic acids

• US 5,354,442 A
• Filed: 05/21/1992
• Issued: 10/11/1994
• Est. Priority Date: 05/21/1992
• Status: Expired due to Fees

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Allen, Robert C., Reeder, Dennis J.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Starsiak, Jr., John S.
• Application Number:
  US07/886,289
• Time in Patent Office: 873 Days
• Field of Search: 204/182.8, 204/299 R
• US Class Current: 204/458
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture

Abstract

The present invention is a method for enhancing the separation of polynucleotides or polypeptides of various chain lengths or a mixture of polynucleotides and polypeptides of various chain lengths using electrophoretic processes to sharpen sample component zones and produce maximum resolution of electrophoretically separated macromolecules by modifying the matrix of electrophoretic gel.

Claims

1. A method of increasing resolution and decreasing mobility of polynucleotides on an electrophoretic support for conducting electrophoresis comprising:

adding to an electrophoretic support for conducting electrophoresis, a polynucleotide resolution increasing concentration of a compound selected from the group consisting of alcohols, polyols, and monosaccharides in the presence of an ionic buffer comprising an ion selected from the group consisting of sulphate, chloride and phosphate ions of a pH of range 6-9, wherein increased resolution and decreased mobility of said polynucleotide on said electrophoretic support results.

2. A method according to claim 1, wherein said ionic buffer is selected from the group consisting of

EPPS (N-2-hydroxyethylpiiperazine-N'"'"'-3-propanesulfonic acid),

HEPPSO (N(2-Hydroxyethyl)piperazine-N'"'"'-(2-hydroxypropanesulfonic acid),

HEPES (N-2-hydroxyethyl)piperazine-N'"'"'-2-ethanesulfonic acid),

BES (N,N-bis-(2-hydroxyethyl)-2-amino-ethanesulfonic acid),

TAPS (3-{(tris-(hydroxymethyl)-methyl)-amino-}-propanesulfonic acid),

TES (3-{tris(hydroxy-methyl)methyl)-amino}-ethanesulfonic acid),

DIPSO (3-(N,N-bis(2-Hydroxyethyl)amino)-2-hydroxypropanesulfonic acid),

MOPS (3-(N-Morpholino) propanesulfonic acid),

MES (2-(N-morpholino)-ethanesulfonic acid),

POPSO (Piperazine-N,N'"'"'-bis-(2-hydroxy-propanesulfonic acid),

PIPES (Piperazine-N,N'"'"'-bis (2-ethanesulfonic acid)),

ACES (N-(2-acetammido)-2-aminoethanesulfonic acid), and

CHES (2-(N-Cyclohexylamino)ethanesulfonic acid).

3. A method according to claim 1, wherein said alcohols are selected from a group consisting of methyl alcohol, glycerol, ethylene glycol, or an ethylene glycol derivative with a hydroxyl group on the number one carbon.

4. A method according to claim 3, wherein said polypeptide resolution modifying concentration of a compound is about 1% to about 20% glycerol.

5. A method according to claim 1, wherein said monosaccharides are selected from the group consisting of sorbose, sorbitol, mannitol, mannose, fructose and ribose with a hydroxyl group on the number-one carbon atom.

6. A method according to claim 1, wherein a polynucleotide to be separated on said electrophoretic support is DNA having from about 5000 to about 10,000 base pairs and electrophoresis used to separate the polynucleotide is pulse constant power electrophoresis.

7. A method according to claim 1, wherein said electrophoresis is constant current electrophoresis.

8. A method according to claim 1, wherein said electrophoresis is constant voltage electrophoresis.

9. A method according to claim 1, wherein said electrophoresis is constant power electrophoresis.

10. A method according to claim 1, wherein said electrophoretic support is an ultra-thin layer polyacrylamide gel.