Detection of nucleic acid heteroduplex molecules by anion-exchange chromatography
OwnerPaul D. Taylorinventor
Life of this patent
Estimating the term…
Abstract
The present invention describes a method for separating or partially separating heteroduplex and homoduplex DNA molecules in a mixture. In the method, the mixture is applied to an anion-exchange chromatography medium. The heteroduplex and homoduplex molecules are eluted with a mobile phase containing an eluting salt, including an anion and a cation, a buffer, and preferably including an organic solvent. The eluting is carried out under conditions effective to at least partially denature the heteroduplexes (e.g., thermal or chemical denaturing) resulting in the separation of the heteroduplexes from the homoduplexes. The method has many applications including, but not limited to, detecting mutations and comparative DNA sequencing.
Claims72
A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a mixture, said method comprising:
- (a) applying the mixture to an anion-exchange solid,
- (b) eluting the solid of step (a) with a mobile phase comprising an eluting salt, an organic solvent, and a buffer, wherein said eluting is carried out under conditions effective to at least partially denature said heteroduplexes and wherein the eluting results in the separation of said heteroduplxes from said homoduplexes.
A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a mixture, comprising:
- (a) applying the mixture to an anion-exchange solid,
- (b) eluting the solid of step (a) with a mobile phase containing an eluting salt and a buffer, where said eluting is carried out under conditions effective to at least partially denature said heteroduplexes and where the eluting results in the separation of said heterodupixes from said homoduplexes.
An aqueous mobile phase useful for the anion-exchange chromatography of double-stranded nucleic acids, said mobile phase comprising:
- an eluting salt in the approximate concentration range of 0.5 to 1.5 M composed of equal concentrations of:
- a cation selected from the group consisting of dialkylammonium, trialkylammonium and tetraalkylammonium wherein the alkyl groups consist of any combination of methyl and ethyl;
- and
- an anion selected from the group consisting of bromide, chloride, acetate, formate, nitrate, perchlorate, dihydrogen phosphate, ethane sulfonate, and methane sulfonate;
- and
- a buffer acid with a pKa in the approximate range of 3.5 to 9.5, which acid has a concentration not exceeding approximately 0.5M, and an organic solvent, which organic solvent has a concentration not exceeding about 40% by volume of said mobile phase.
An aqueous mobile phase useful for the anion-exchange chromatography of double-stranded nucleic acids, said mobile phase comprising:
- an eluting salt in the approximate concentration range of 1.0M to 2.0M composed of equal concentrations of:
- a cation selected from the group consisting of dialkylammonium, trialkylammonium and tetraalkylammonium wherein the alkyl groups consist of any combination of methyl and ethyl;
- and
- an anion selected from the group consisting of bromide, chloride, acetate, formate, nitrate, perchlorate, dihydrogen phosphate, ethane sulfonate, and methane sulfonate;
- and
- a buffer acid with a pKa in the approximate range of 3.5 to 9.5, which acid has a concentration not exceeding approximately 0.5M;
- an organic solvent, which organic solvent has a concentration not exceeding about 40% by volume of said mobile phase.
A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a mixture, said method comprising:
- (a) applying the mixture to an anion-exchange solid,
- (b) eluting the solid of step (a) with a mobile phase containing an eluting salt, an organic solvent, and a buffer, wherein said eluting is carried out under conditions effective to at least partially denature said heteroduplexes and wherein the eluting results in the separation of said heteroduplxes from said homoduplexes;
- wherein step (b) includes contacting the solid of step (a) with a mobile phase possessing a pH in the range of 4 to 9 comprising:
- an eluting salt comprising equal concentrations of:
- a cation;
- an anion;
- a buffer acid with a pKa in the approximate range of 3.5 to 9.5;
- and,
- an organic solvent;
- wherein said mobile phase contains less than about 40% by volume of said organic solvent;
- wherein the concentration of eluting salt is systematically increased from approximately 0.5M to approximately 2.0M.
A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a mixture, comprising:
- (a) applying the mixture to an anion-exchange solid,
- (b) eluting the solid of step (a) with a mobile phase containing an eluting salt, an organic solvent, and a buffer, wherein said eluting is carried out under conditions effective to at least partially denature said heteroduplexes and wherein the eluting results in the separation of said heteroduplxes from said homoduplexes;
- wherein step (b) includes contacting the solid of step (a) with a mobile phase possessing a pH in the range of 4 to 9 comprising:
- an eluting salt comprising:
- betaine at a concentration in the range of about 0.5M to about 6M;
- a buffer acid with a pKa in the approximate range of 3.5 to 9.5;
- and,
- an organic solvent;
- wherein said mobile phase contains less than about 40% by volume of said organic solvent;
- wherein the concentration of eluting salt is systematically increased from approximately 0.5M to approximately 2.0M.
A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a mixture, said method comprising:
- (a) applying the mixture to an anion-exchange solid,
- (b) eluting the solid of step (a) with a mobile phase containing an eluting salt, an organic solvent, and a buffer, where said eluting is carried out under conditions effective to at least partially denature said heteroduplexes and where the eluting results in the separation of said heterodupixes from said homoduplexes;
- wherein step (b) includes contacting the solid of step (a) with a mobile phase possessing a pH in the range of 4 to 9 comprising:
- an eluting salt comprising equal concentrations of:
- a cation;
- an anion;
- a buffer acid with a pKa in the approximate range of 3.5 to 9.5;
- and, wherein the eluting is carried out at a column temperature greater than about 50° C., wherein the concentration of eluting salt is systematically increased from approximately 0.5M to approximately 2.0M.
A method for detecting DNA genetic mutations, the method comprising:
- a) heating a mixture of a sample double stranded DNA segment and a corresponding wild type double stranded DNA segment to a temperature at which the strands are completely denatured;
- b) cooling the product of step (a) until the strands are completely annealed, whereby a mixture comprising two homoduplexes and two heteroduplexes is formed if the sample segment includes a mutation;
- c) determining the heteromutant site separation temperature;
- d) analyzing the product of step (b) with Denaturing Anion-Exchange High Performance Chromatography at the heteromutant site separation temperature to identify the presence of any heteromutant site separated components therein.
Dependencies are inferred from the claim text.