System and method for automated matched ion polynucleotide chromatography

A high pressure liquid chromatography system including a computerized control means, the chromatography system comprising mobile phase flow control means for controlling the flow of solvent solution and aqueous components of a mobile phase, the flow control means including computer instruction input means;

• a Matched Ion Polynucleotide Chromatography separation column;
• conduit means for directing mobile phase from the mobile phase flow control means to the separation column;
• and a computer in communication with the mobile phase flow control means;
• a software mobile phase flow control module in working association with the computer and the mobile phase flow control means, wherein the mobile phase flow control means are a set of flow control valves, each with automatic opening controls responsive to control commands from the flow control module or the mobile phase control means is a set of pumps, the flow setting of which are responsive to control commands from the flow control module, said computer including solvent concentration and gradient computing software for computing the solvent gradient beginning and ending solvent concentrations for the separation of double-stranded DNA fragments.

In a method for effecting a separation of equal length heteroduplex and homoduplex DNA molecules in a mixture, with a high pressure liquid chromatography system comprising a Matched Ion Polynucleotide Chromatography separation column;

• oven temperature control means for controlling the temperature of mobile phase entering the column, the oven temperature control means including computer instruction input means;
• and a computer in communication with the oven temperature control means having a oven temperature control software module resident therein;
• the method comprising the computerized steps of:
• analyzing the mixture by Denaturing Matched Ion Polynucleotide chromatography in a series of Denaturing Matched ion Polynucleotide Chromatography separations in the temperature range of about 50° C. to about 75° C., each successive separation having a higher temperature than the preceding separation until a mutation separation profile is observed or the absence of any mutation separation profile in the mutation separation temperature range is observed, wherein a mutation separation profile identifies the presence of a mutation and the absence of a mutation separation profile indicates an absence of mutation in the sample;
• and
• wherein the oven temperature control module controls the settings of the oven temperature control means to effect the temperature during the successive separations.

In a method for effecting a separation of equal length heteroduplex and homoduplex DNA molecules in a mixture, with a high pressure liquid chromatography system comprising a Matched Ion Polynucleotide Chromatography separation column;

• oven temperature control means for controlling the temperature of mobile phase entering the column, the oven temperature control means including computer instruction input means;
• and a computer in communication with the oven temperature control means having a oven temperature control software module resident therein;
• the method comprising the computerized steps of:
• analyzing the mixture by Denaturing Matched Ion Polynucleotide Chromatography in a series of Denaturing Matched Ion Polynucleotide Chromatography separations in the temperature range of 50° C. to about 75° C., each successive separation having a lower temperature than the preceding separation until a mutation separation profile is observed or the absence of any mutation separation profile in the mutation separation temperature range is observed, wherein a mutation separation profile identifies the presence of a mutation and the absence of a mutation separation profile indicates an absence of mutation in the sample;
• and
• wherein the oven temperature control module controls the settings of the oven temperature control means to effect the temperature during the successive separations.

In a method for effecting a separation of equal length heteroduplex and homoduplex DNA molecules in a mixture, with a high pressure liquid chromatography system comprising a Matched Ion Polynucleotide Chromatography separation column;

• oven temperature control means for controlling the temperature of mobile phase entering the separation column, the oven temperature control means including computer instruction input means;
• and a computer in communication with the oven temperature control means having a oven temperature control software module resident therein;
• the method comprising the computerized steps of:
• a) receiving a predicted heteromutant site separation temperature;
• b) heating said mixture of DNA molecules to said predicted heteromutant site separation temperature;
• and
• c) analyzing the product of step (b) with Denaturing Matched Ion Polynucleotide Chromatography at the predicted heteromutant site separation temperature to identify the presence of any heteromutant site separated components therein.

A denaturing high pressure liquid chromatography system including a computerized control means, the chromatography system comprising a Matched Ion Polynucleotide Chromatography separation column;

• oven temperature control means for controlling the temperature of the mobile phase entering the separation column, the temperature control means including computer instruction input means;
• conduit means for directing mobile phase the MIPC separation column;
• a computer in communication with the oven temperature control means;
• a oven temperature control software module in working association with the computer and the oven temperature control means, said computer including temperature computing software for computing the heteromutant site separation temperature for the separation of equal length heteroduplex and homoduplex DNA molecules in a mixture.

In a method for effecting a separation of equal length heteroduplex and homoduplex DNA molecules in a mixture, with a high pressure liquid chromatography system comprising mobile phase flow control means for controlling the flow of solvent solution and aqueous components of a mobile phase, the flow control means including computer instruction input means;

• a Matched Ion Polynucleotide Chromatography separation column;
• conduit means for directing mobile phase from the mobile phase flow control means to the separation column;
• and a computer in communication with the mobile phase flow control means having a software mobile phase flow control module resident therein;
• the computerized steps of:
• a) receiving the numerical value of the base-pair length of a double-stranded DNA molecule having the length of a homoduplex or heteroduplex molecule in the mixture;
• b) calculating a solvent concentration which will effect elution of a molecule having said base-pair length from said column under non-denaturing conditions, wherein said calculating comprises using an algorithm for calibrating said chromatography system and said column, wherein said algorithm relates the base pair length of DNA fragments in a standard mixture of double-stranded DNA fragments to solvent concentration required to elute the fragments in said standard mixture;
• c) calculating a range of solvent concentrations and corresponding mobile phase gradient to begin below a solvent concentration that will elute said double-stranded DNA molecule and to end above a solvent concentration that will elute said double-stranded DNA molecule;
• d) providing said range of solvent concentrations and corresponding mobile phase gradient to the mobile phase flow control module;
• and
• e) conducting the separation under conditions effective to partially denature said heteroduplexes, wherein the mobile phase flow control module controls the settings of the mobile phase flow control means to effect said range of solvent concentrations and corresponding mobile phase gradient to effect separation of said homoduplex and said heteroduplex molecules.

A denaturing high pressure liquid chromatography system including a computerized control means, the chromatography system comprising mobile phase flow control means for controlling the flow of solvent solution and aqueous components of a mobile phase, the flow control means including computer instruction input means;

• a Matched Ion Polynucleotide Chromatography separation column;
• conduit means for directing mobile phase from the mobile phase flow control means to the separation column;
• and a computer in communication with the mobile phase flow control means;
• a software mobile phase flow control module in working association with the computer and the mobile phase flow control means, wherein the mobile phase flow control means are a set of flow control valves, each with automatic opening controls responsive to control commands from the flow control module or the mobile phase control means is a set of pumps, the flow setting of which are responsive to control commands from the flow control module, said computer including solvent concentration and gradient computing software for computing the solvent gradient and beginning and ending solvent concentrations for the separation of equal length heteroduplex and homoduplex DNA molecules in a mixture.

In a method for effecting a separation of equal length heteroduplex and homoduplex DNA molecules in a mixture, with a high pressure liquid chromatography system comprising a Matched Ion Polynucleotide Chromatography separation column;

• mobile phase flow control means for controlling the flow of solvent solution and aqueous components of a mobile phase, the flow control means including computer instruction input means;
• conduit means for directing mobile phase from the mobile phase flow control means to the separation column;
• oven temperature control means for controlling the temperature of mobile phase entering the separation column, the oven temperature control means including computer instruction input means;
• and a computer in communication with the mobile phase flow control means having a software mobile phase flow control module resident therein;
• said computer in communication with the oven temperature control means having a oven temperature control software module resident therein;
• the method comprising the computerized steps of:
• a) receiving the numerical value of the base-pair length of a double-stranded DNA molecule having the length of a homoduplex or heteroduplex molecule in the mixture;
• b) calculating a %B which will effect elution of a double-stranded DNA molecule having said base-pair length from said column under non-denaturing conditions, wherein said calculating comprises using an algorithm for calibrating said chromatography system and said column, wherein said algorithm relates the base pair length of a standard mixture of double-stranded DNA fragments to the %B required to elute the fragments in said standard mixture, wherein %B is the percentage in the mobile phase of an aqueous solution containing organic solvent;
• c) calculating a range of %B and corresponding mobile phase gradient to begin below the value of %B that will elute said double-stranded DNA molecule having said base-pair length and to end above a %B that will elute said double-stranded DNA molecule having said base-pair length;
• d) providing said range of %B and corresponding mobile phase gradient to the mobile phase flow control module;
• e) receiving a predicted heteromutant site separation temperature;
• heating said mixture of DNA molecules to said predicted heteromutant site separation temperature;
• and
• f) conducting the separation, wherein the mobile phase flow control module controls the settings of the mobile phase flow control means to effect said range of %B and corresponding mobile phase gradient required to effect separation of the homoduplex and heteroduplex fragments by Denaturing Matched Ion Polynucleotide Chromatography at the predicted heteromutant site separation temperature to identify the presence of any heteromutant site separated components in the mixture.