Nucleic acid separation using immobilized metal affinity chromatography

a technology of immobilized metal and affinity chromatography, which is applied in the direction of sugar derivates, organic chemistry, chemistry apparatus and processes, etc., can solve the problem that imac has found very limited application in the purification of nucleic acids

Inactive Publication Date: 2011-10-20
WILLSON RICHARD C +1
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Benefits of technology

[0032]The present invention also provides a plasmid separation technique using membranes coated with or impregnated with an IMAC matrix to filter out or prevent free RNA from migrating to the other side of the membrane structure.

Problems solved by technology

While metal ion binding to nucleic acids is well known, and plays an important role in the function of the widely used cancer drug cisplatin (10), IMAC has found very limited application in the purification of nucleic acids.

Method used

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  • Nucleic acid separation using immobilized metal affinity chromatography
  • Nucleic acid separation using immobilized metal affinity chromatography
  • Nucleic acid separation using immobilized metal affinity chromatography

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[0205]Equilibrium Adsorption Isotherms 1

[0206]To test binding capacities and trends, equilibrium adsorption isotherms were run to find affinities of RNA and DNA to IMAC matrices. The first experiment ran was to distinguish the different binding affinities of RNA and double-stranded DNA. In the example, equilibrium isotherms were performed using Zn (II) charged IDA IMAC resin using total bakers yeast RNA and pBGS19Luxwt plasmid DNA. The resulting isotherms showed no apparent binding of the plasmid DNA, but a defined binding isotherm for bakers yeast RNA. This experiment represented was the first evidence of differential binding affinity to an IMAC reagent for single-stranded RNA and double-stranded DNA.

[0207]Next, binding isotherms were run using different divalent metal ions (Cu.sup.2+, Ni.sup.2+, Zn.sup.2+ and Co.sup.2+) and total bakers yeast RNA to determine the relative binding efficiencies. FIG. 1 shows the isotherms for each divalent metal ion. From the isotherms is it apparen...

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Abstract

An immobilized metal affinity chromatography (IMAC) method for separating and / or purifying compounds containing a non-shielded purine or pyrimidine moiety or group such as nucleic acid, presumably through interaction with the abundant aromatic nitrogen atoms in the purine or pyrimidine moiety. The method can also be used to purify compounds containing purine or pyrimidine moieties where the purine and pyrimidine moieties are shielded from interaction with the column matrix from compounds containing a non-shielded purine or pyrimidine moiety or group. Thus, double-stranded plasmid and genomic DNA, which has no low binding affinity can be easily separated from RNA and / or oligonucleotides which bind strongly to metal-charged chelating matrices. IMAC columns clarify plasmid DNA from bacterial alkaline lysates, purify a ribozyme, and remove primers and other contaminants from PCR reactions. The metal ion affinity of yeast RNA decreases in the order: copper (II), nickel (II), zinc (II), and cobalt (II).

Description

RELATED APPLICATIONS[0001]This application is a Divisional of and claims priority of U.S. patent application Ser. No. 09 / 994,701 filed Nov. 6, 2001 now U.S. Pat. No. 7,598,371, which claims priority of U.S. Provisional Patent Application Ser. No. 60 / 246,292 filed Nov. 6, 2000.RELATED APPLICATIONS[0002]This application claims provisional priority to U.S. Provisional Patent Application Ser. No. 60 / 246,292 filed Nov. 6, 2000.GOVERNMENT LICENSE RIGHTS STATEMENT[0003]The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of grant No. R825354-01-0 from the National Space Biomedical Research Institute and the Environmental Protection Agency.BACKGROUND OF THE INVENTION[0004]1. Field of the Invention[0005]The present invention relates to an immobilized metal affinity chromatography (IMAC) instrument, a substrate containing immobilized metal affinity ligan...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H1/06C12N15/10
CPCC12N15/101
Inventor WILLSON, RICHARD C.MURPHY, JASON C.
Owner WILLSON RICHARD C
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