Solid phase technique for selectively isolating nucleic acids

A method of separating host cell DNA from exogenous DNA present in a host cell comprising the steps of:

• (a) combining a host cell lysate with:
• (1) paramagnetic microparticles with a functional group-coated surface, (2) polyethylene glycol and (3) a salt, wherein polyethylene glycol and the salt are present in concentrations which result in binding of host cell DNA to the microparticles, but not in binding of exogenous DNA to the microparticles, thereby producing a first combination, which comprises host cell DNA bound to the microparticles and unbound exogenous DNA;
• (b) separating the paramagnetic microparticles from the first combination, thereby producing a mixture that is enriched for exogenous DNA;
• (c) adding to the mixture paramagnetic microparticles with a functional group-coated surface and polyethylene glycol at a concentration that results in binding of exogenous DNA to the microparticles, thereby producing a second combination, which comprises exogenous DNA bound to microparticles;
• and
• (d) separating the paramagnetic microparticles from the second combination, whereby exogenous DNA is separated from host cell DNA.

A method of separating plasmid DNA from high molecular weight DNA present in a host cell lysate comprising:

• (a) preparing a suspension comprising carboxyl group-coated paramagnetic microparticles and a host cell lysate, wherein the host cell lysate comprises lysed host cells, low percentage PEG, and low molarity salt, wherein the PEG and salt are present in sufficient concentrations that high molecular weight DNA binds to the carboxyl group-coated paramagnetic microparticles, thereby producing a first combination comprising suspended paramagnetic microparticles having high molecular weight DNA bound thereto and unbound plasmid DNA;
• (b) removing the paramagnetic microparticles from the first combination, thereby removing high molecular weight DNA from the first combination and producing a plasmid-enriched supernatant;
• (c) adding to the plasmid-enriched supernatant carboxyl group-coated paramagnetic microparticles and sufficient polyethylene glycol to result in the binding of plasmid DNA to the paramagnetic microparticles, thereby producing a second combination;
• and
• (d) removing the paramagnetic microparticles having plasmid DNA bound thereto from the second combination,
• thereby separating plasmid DNA from high molecular weight DNA present in a host cell lysate.

An automatable method of producing plasmid DNA suitable for use as a template for nucleotide sequencing comprising the steps of:

• (a) treating a host cell with an alkali and anionic detergent combination, thereby producing a lysed host cell suspension;
• (b) combining the lysed host cell suspension with paramagnetic microparticles which bind DNA in the lysed host cell suspension with low percentage polyethylene glycol and low molarity salt, whereby host cell DNA is precipitated and binds to paramagnetic microparticles, thereby producing a first combination;
• (c) removing paramagnetic microparticles having host cell DNA bound thereto from the first combination, thereby producing a plasmid-enriched supernatant;
• (d) combining paramagnetic microparticles which bind DNA with the plasmid-enriched supernatant of (c), wherein the polyethylene glycol percentage and salt concentration are sufficient to result in binding of plasmid DNA to the paramagnetic microparticles, thereby producing a second combination;
• (e) removing paramagnetic microparticles having plasmid DNA bound thereto from the second combination;
• (f) washing the paramagnetic microparticles having plasmid DNA bound thereto with a wash buffer that dissolves impurities adsorbed to the paramagnetic microparticles but does not solubilize the plasmid DNA bound to the paramagnetic microparticles, thereby removing impurities adsorbed to the paramagnetic microparticles;
• and
• (g) contacting the paramagnetic microparticles of (f) with an elution buffer suitable for nucleotide sequencing, whereby plasmid DNA is released from the microparticles and dissolved in the elution buffer, thereby producing plasmid DNA suitable for use as a template for nucleotide sequencing.

A kit for high through-put automated DNA template production comprising:

• (a) reagents for preparing a host cell lysate;
• (b) an aqueous solution of functional group-coated paramagnetic microparticles;
• and
• (c) at least one binding buffer formulated to comprise a suitable salt and a suitable polyethylene glycol, wherein the salt and the polyethylene glycol are each present at a concentration appropriate for binding a nucleic acid species characterized by a particular molecular size to the microparticles or reagents for the formulation of a suitable buffer.

A method of selectively isolating extension products from a sequencing reaction mixture, comprising the steps of:

• (a) preparing a combination comprising (1) an aliquot of labeled energy transfer dye terminator or dye primer nucleotide sequencing reaction products comprising extension products;
• (2) carboxyl-coated paramagnetic microparticles and (3) a nucleic acid precipitating agent, wherein the precipitating reagent is present in sufficient concentration to selectively precipitate the extension products, but not other nucleic acid having a molecular size which is smaller than the molecular size of the extension products in the reaction mixture;
• (b) maintaining the combination under conditions appropriate for adsorption of the precipitated extension products to the functional group-coated surface of the microparticles, thereby producing extension product-coated microparticles;
• (c) removing the extension product-coated microparticles from the combination;
• (d) eluting the extension products from the microparticles;
• and
• (e) removing the solid phase carriers from the solution of (e), whereby the extension products of a sequencing reaction mixture are selectively isolated.

A method of selectively isolating a target species of nucleic acid molecules present in a mixture of nucleic acid molecules of different molecular sizes, wherein the target species is of a particular molecular size, said method comprising the steps of:

• (a) preparing a combination comprising the mixture of nucleic acid molecules, polyethylene glycol, salt, and solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules, wherein the polyethylene glycol and salt are present in sufficient concentrations to selectively precipitate the target species of nucleic acid molecule, but not other species of nucleic acid molecules having a molecular size which is less than the molecular size of the target species, thereby producing a first combination;
• (b) maintaining the first combination under conditions appropriate for adsorption of the precipitated nucleic acid molecules to the functional group-coated surface of the solid phase carriers, thereby producing solid phase carriers having bound thereto the target species of nucleic acid molecules;
• (c) removing the carriers having bound thereto the target species of nucleic acid molecules from the first combination, thereby producing a second mixture comprising the nucleic acid molecules having a molecular size which is less than the molecular size of the target species;
• and
• (d) eluting the target species of nucleic acid molecules from the carriers.