Methods for separating nucleic acids with graphene coated magnetic beads

Abstract

Methods for separating and identifying nucleic acids utilize carbon coated magnetic beads. The method teaches that multivalent cations promote binding of single stranded nucleic acids to the beads and that the single stranded nucleic acids can be released with the addition of chelating agents that bind the multivalent cations such as EDTA. The method further teaches that fragile single stranded nucleic acids, such as RNA, can be stored on the surface of the beads. Lastly, the method also teaches that by iteratively adding complimentary DNA oligos, single stranded nucleic acids can be quantified or individually isolated using the carbon coated magnetic beads.

1 . A method for separating single stranded nucleic acids from associated biological material, the method comprising:
 creating a complex of a carbon coated material and single stranded nucleic acids from a mixture containing single stranded nucleic acids, double stranded nucleic acids, the carbon coated material, at least one chaotropic salt and at least one alcohol having between one and four carbon atoms, wherein the material is a chaotropic salt present at a concentration from 0.01M to 3M and wherein the alcohols are present as 5 vol % to 99 vol %; and   after the complex of carbon coated material and single stranded nucleic acids has been created, removing the carbon coated material complexed with single-stranded nucleic acid from the mixture.   
     
     
         2 . The method of  claim 1  wherein the carbon coating present on the carbon coated material is at least one of graphene, pyrolytic carbon or a mixture of graphene and pyrolytic carbon. 
     
     
         3 . The method of  claim 1 , wherein chaotropic salt is a guanidinium salt composed of a guanidinium cation and an anion selected from the group consisting of chloride, bromide, fluoride, acetate, sulfate, nitrate, carbonate, thiocyanate and mixtures thereon. 
     
     
         4 . The method of  claim 1 , wherein the chaotropic salt is present at a concentration between 0.01 M and 0.6M. 
     
     
         5 . The method of  claim 1 , wherein the alcohol is present in an amount between 10 vol % and 85 vol %. 
     
     
         6 . A method for isolating nucleic acids having a defined sequence from an aqueous solution, the method comprising:
 contacting a composition containing single stranded nucleic acid and double stranded nucleic acid with at least one nucleic acid probe, the nucleic acid probe complimentary with amino acids present on at least a portion of the nucleic acid in the aqueous solution, the contacting step occurring for an interval sufficient for the at least one nucleic acid probe to duplex with amino acid sequences present on at least a portion of the nucleic acid present in the solution to produce an admixture composed of duplexed nucleic acid material and non-duplexed nucleic acid material;   to the resulting admixture containing duplexed nucleic acid material, adding carbon-coated material, at least one chaotropic salt and short-chain alcohol, the short-chain alcohol present in an amount between 2 vol % and 99 vol %;   allowing contact for an interval sufficient to permit association between the carbon coated material and at least a portion of the single stranded nucleic acids present in the admixture;   separating carbon-coated material complexed with the single stranded nucleic acids from liquid supernatant, the liquid supernatant containing complexed double stranded nucleic acid.   
     
     
         7 . The method of  claim 6 , wherein the carbon coated material is configured as beads, the beads each having a core and an outer surface wherein at least the core is magnetic and contains at least 30% by weight of a metal selected from the group consisting of Ni, Fe, Co, or mixtures thereof. 
     
     
         8 . The method of  claim 6 , wherein the chaotropic salt is added in an amount sufficient to provide a concentration between 0.01M and 3M. 
     
     
         9 . The method of  claim 8 , wherein chaotropic salt is selected from the group consisting of a guanidinium salts, urea, thiourea, lithium acetate, lithium perchlorate, magnesium chloride and mixtures thereof. 
     
     
         10 . The method of  claim 9 , wherein chaotropic salt is a guanidinium salt composed of a guanidinium cation and an anion selected from the group consisting of chloride, bromide, fluoride, acetate, sulfate, nitrate, carbonate, thiocyanate and mixtures thereof. 
     
     
         11 . The method of  claim 10 , wherein the concentration of the chaotropic salt is between 0.01 and 8M. 
     
     
         12 . The method of  claim 6 , wherein the short chain alcohol is selected from the group consisting of methanol, ethanol, propanol, isopronaol, phenol, and mixtures thereof. 
     
     
         13 . The method of  claim 6 , wherein the removing step includes subjecting the resulting complex of carbon-coated magnetic beads and nucleic acid to at least one of a magnetic field, a centrifugal force, precipitation, competitive binding, or mixtures thereof. 
     
     
         14 . The method of  claim 6 , wherein the concentration of alcohols is between 10% and 95%. 
     
     
         15 . The method of  claim 6 , wherein the carbon coated material comprises a substrate and an outer carbon coating, the carbon coating having a thickness between 1 angstrom and 50 nm. 
     
     
         16 . The method of  claim 6 , further comprising:
 after removing the carbon coated magnetic beads from the supernatant, releasing single-stranded nucleic acid from attachment to the carbon coated material, wherein the releasing step occurs with addition of water or suitable buffer solution into contact with the removed carbon coated material.   
     
     
         17 . The method of  claim 16  wherein the removal step includes at least one of the following: centrifugation, precipitation, competitive binding, subjecting the composition to a magnetic field. 
     
     
         18 . The method of  claim 6 , wherein the carbon-coated material is configured as individual beads, each bead having an interior core and an outer surface, the beads composed of one of the following: a magnetic metal containing at 30% by weight of a metal selected from the group consisting of Ni, Fe, Co, or mixtures thereof, silica or polymeric substrates, and wherein the carbon coating is present as a carbon layer on at least a portion of the outer surface of the bead, the carbon layer comprising at least one of graphene, pyrolytic carbon or a mixture of graphene and pyrolytic carbon, and wherein the multivalent cations are alkali earth metals or alkali earth metal salts or mixtures thereof. 
     
     
         19 . The method of  claim 6 , wherein the complementary nucleic acid probe is a chain of nucleic acids selected such that the chain of nucleic acids contains complimentary bases with corresponding positions to the single stranded nucleic acid that is to be isolated or purified. 
     
     
         20 . The method of  claim 6 , wherein the complementary nucleic acid probe may be DNA or a synthetic nucleic acid and wherein the method further comprises the step of quantifying the sequence for study by performing PCR on the supernatant which remains after removal of carbon coated magnetic material and associated single stranded nucleic acids.