Nucleic acid separation using immobilized metal affinity chromatography

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).

1 . A method for separating compounds comprising the steps of: contacting a mixture comprising cell lysate or enzyme and a target polynucleotide compound which includes at least four shielded purine or pyrimidine moieties, and at least one contaminant polynucleotide compound which includes at least four non-shielded purine or pyrimidine moieties, and other compounds, with a solid composition including immobilized metal ions capable of binding compounds containing a non-shielded purine or pyrimidine moiety, to form a liquid product containing a reduced amount of the contaminant polynucleotide and also at least 10% of the original amount of the target polynucleotide compound which includes at least four shielded purine or pyrimidine moieties, substantially free from histidine tags. 
     
     
         2 . The method of  claim 1  in which the solid composition comprises a polymeric material including a plurality of ligands bonded thereto, each ligand immobilizing a metal ion selected from the group consisting of Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+, Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ or mixtures or combinations thereof. 
     
     
         3 . A method for separating compounds comprising the steps of: contacting a mixture comprising cell lysate or enzyme and a target protein, and a contaminant compound which includes at least four non-shielded purine or pyrimidine moieties, and other compounds, with a solid composition including immobilized metal ions capable of binding compounds containing a non-shielded purine or pyrimidine moiety, to form a liquid product containing a reduced amount of the contaminant polynucleotide; and collecting the target protein substantially free of polynucleotides. 
     
     
         4 . A method according to  claim 3  comprising collecting the target protein with a reduced content of polynucleotides. 
     
     
         5 . A method according to  claim 3  comprising collecting the target protein substantially free of genomic DNA. 
     
     
         6 . A method according to  claim 3  comprising collecting the target protein substantially free of RNA. 
     
     
         7 . The method of  claim 3  in which the solid composition comprises a polymeric material including a plurality of ligands bonded thereto, each ligand immobilizing a metal ion selected from the group consisting of Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+, Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ or mixtures or combinations thereof. 
     
     
         8 . A substrate comprising a plurality of ligands bonded thereto, each ligand immobilizing a metal atom and/or ion capable of binding compounds containing at least 4 non-shielded purine or pyrimidine moieties or groups, and a compound containing at least 4 non-shielded purine or pyrimidine moieties or groups bound to a portion of the metal atoms and/or ions wherein the compound is selected from the group of RNA, single stranded DNA, and other molecules having at least 4 non-shielded purine and/or pyrimidine moieties or groups; or wherein the substrate comprises a polymeric material including a plurality of ligands bonded thereto, each ligand immobilizing a metal ion selected from the group consisting of Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Fe.sup.3+, Fe.sup.2+, Co.sup.2+, Sc.sup.2+, Ti.sup.2+, V.sup.2+, Cr.sup.2+, Mn.sup.2+, Ca.sup.2+, Cd.sup.2+ or Hg.sup.2+ or mixtures or combinations thereof; or wherein the substrate is selected from the group consisting of a polymer, a column inner wall, a membrane, an inorganic support, a metallic support, and a surface of an electronic chip. 
     
     
         9 . The method of  claim 1  further comprising the step of: separating the liquid from the solid composition. 
     
     
         10 . A method according to  claim 1  further comprising the steps of: separating the supernatant liquid from the solid composition; or further comprising the steps of: separating the supernatant liquid from the solid composition and eluting the RNA and/or DNA including a non-shielded purine or pyrimidine moiety from the solid composition. 
     
     
         11 . Method according to  claim 1  wherein the mixture comprises poly(A) tailed mRNA sequences and other mRNA sequences from eukaryotic cells, or wherein the mixture of compounds comprises denatured nucleic acid sequences, wherein sequences having A-rich regions bind better than sequences having T-rich regions, so that complementary strands can be resolved. 
     
     
         12 . A method of  claim 1  wherein the solid phase comprises a well plate, particle, magnetic particle, tube, pipette tip, monolith, or chromatographic packing. 
     
     
         13 . The method of  claim 1  wherein the contaminant compound comprises RNA having at least four non-shielded purine and/or pyrimidine moieties and is separated from a lysate containing double-stranded DNA. 
     
     
         14 . The method of  claim 1  wherein the contaminant compound comprises DNA having at least four non-shielded purine and/or pyrimidine moieties and is separated from a mixture containing double-stranded DNA and an enzyme. 
     
     
         15 . The method of  claim 1  wherein the target polynucleotide compound which includes at least four shielded purine or pyrimidine moieties is present in the original mixture at a concentration of less than 1 micromolar. 
     
     
         16 . A method of  claim 1  wherein the target compound comprises DNA. 
     
     
         17 . A method of claim  110  wherein the contaminant compound comprises RNA. 
     
     
         18 . The method of  claim 3  wherein the target protein is present in the original mixture at a concentration of less than 1 micromolar. 
     
     
         19 . The method of  claim 3  further comprising the step of: separating the liquid from the solid composition. 
     
     
         20 . A method according to  claim 3  further comprising the steps of: separating the supernatant liquid from the solid composition; or further comprising the steps of: separating the supernatant liquid from the solid composition and eluting the RNA and/or DNA including a non-shielded purine or pyrimidine moiety from the solid composition.