Apparatus and method for separating and purifying polynucleotides

A method for determining the presence of DNA fragments having a specific base-pair length in a sample mixture, the method comprising:

• a) applying the sample mixture to a separation column containing separation media having a nonpolar, nonporous surface, the sample mixture being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) removing any DNA in the sample having said specific base pair length by contacting the separating media with a second solvent solution containing a counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having said base pair from the separation media.

A method for separating, by Denaturing Matched Ion Polynucleotide Chromatography, target homoduplex and/or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.

A method for determining the presence of a homoduplex or a heteroduplex fraction in a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.

A method for isolating DNA fragments in a mixture, the method comprising:

• a) applying the mixture of DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solution containing counterion and lacking driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) removing the DNA fragments from the media by contacting the media with a second solution containing counterion and a concentration of driving solvent comprising acetonitile at a fixed concentration of at least about 25%.

A method for determining the presence of a homoduplex or a heteroduplex fraction in a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.

A method for separating, by Denaturing Matched Ion Polynucleotide Chromatography, target homoduplex and/or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.

A method for determining the presence of a homoduplex or a heteroduplex fraction in a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions, wherein the concentration of driving solvent in the second solution was previously determined from a reference standard.

A method for separating, by Denaturing Matched Ion Polynucleotide Chromatography, target homoduplex and/or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site thereof, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions, wherein the concentration of driving solvent in the second solution was previously determined from a reference standard.

A method for isolating targeted DNA fragments having a predetermined base-pair length from a mixture of DNA fragments, the method comprising:

• a) applying the mixture of DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solution containing counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) removing the targeted DNA fragments from the media by contacting the media with a second solution containing counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having the targeted fragments from the separation media into a distinct segment of eluant.

A method for isolating DNA fragments in a mixture, the method comprising:

• a) applying the mixture of DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solution containing counterion and lacking driving solvent, wherein said media comprises silica, and wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) removing the DNA fragments from the media by contacting the media with a second solution containing counterion and a concentration of driving solvent comprising acetonitile at a fixed concentration of at least about 50%.

A method for isolating targeted DNA fragments having a predetermined base-pair length from a mixture of DNA fragments, the method comprising:

• a) applying the mixture of DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solution containing counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) removing the targeted DNA fragments from the media by contacting the media, under ambient or low pressure, with a second solution containing counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having the targeted fragments from the separation media into a distinct segment of eluant.

A method for determining the presence of DNA fragments having a specific base-pair length in a sample mixture, the method comprising:

• a) applying the sample mixture to a separation column containing separation media having a nonpolar, nonporous surface, the sample mixture being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) removing any DNA in the sample having said specific base pair length by contacting, under ambient or low pressure, the separating media with a second solvent solution containing a counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having said base pair from the separation media.

A method for separating target homoduplex and/or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting, under ambient or low pressure, the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.

A method for determining the presence of a homoduplex or a heteroduplex fraction in a mixture of homoduplex and heteroduplex DNA fragments having the same base-pair length, the heteroduplex fragments having at least one mismatch site, the method comprising the steps of:

• a) applying the mixture of homoduplex and heteroduplex DNA fragments to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of homoduplex and heteroduplex DNA fragments being applied in a first solution containing a counterion and a DNA binding concentration of driving solvent, wherein said surface is substantially free of multivalent cations capable of binding DNA;
• and
• b) while maintaining the media and the solution at a temperature which will locally denature the heteroduplex fragments at the mismatch site of the heteroduplex fragments, separating desired homoduplex and/or heteroduplex DNA fragments from the separation media by contacting, under ambient or low pressure, the separation media with a second solution containing a counterion and a concentration of driving solvent which has been predetermined to separate the target homoduplex and/or heteroduplex DNA fragments from the separation media in separate fractions.