Methods and compositions for mutation analysis of polynucleotides by liquid chromatography

A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a mixture, said method comprising:

• (a) applying the mixture to a reverse phase separation medium;
• (b) eluting the medium of step (a) with a mobile phase comprising a nitrogen-containing additive, 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 heteroduplexes from said homoduplexes,
• wherein said eluting is carried out at a pre-selected concentration of said nitrogen-containing additive and a at pre-selected temperature;
• wherein said additive is present at a concentration at which first heteroduplex molecules from a first mixture can be observed at said pre-selected temperature, and wherein second heteroduplex molecules from a second mixture can be observed at said pre-selected temperature;
• wherein said first mixture comprises a hybridization product of the double stranded sequence variant 168A and sequence variant 168G of a first fragment, wherein the 168A variant corresponds to the nucleic acid identified by SEQ ID NO:
• 1;
• and,
• wherein said second mixture comprises a hybridization product of the double stranded sequence variant 46C and sequence variant 46G of a second fragment, wherein the 46C variant corresponds to the nucleic acid identified by SEQ ID NO:
• 2;
• wherein said nitrogen-containing additive comprises a compound according to the formula:
• 7
• wherein:
• R1, R2, and R3, may be the same or different and are independently selected from the group consisting of hydrogen, methyl, ethyl, hydroxyethyl, and propyl, with the proviso that no more than two of R1, R2, and R3 are hydrogen;
• and
• X is a moiety selected from the group consisting of:
• radicals of the formulas
• ═O →O —CH3;
• —CH2CH3;
• and
• 8
• wherein:
• R4 is selected from the group consisting of methyl and hydrogen and, when combined with R1 , forms a pyrrolidine ring;
• R5 is selected from the group consisting of —CO2H, —CH2OH, and —SO3H;
• and
• n is an integer of from 0 to 2;
• with the proviso that, when R1 and R4 form a pyrrolidine ring, no more than one of R2 and R3 is hydrogen.

An aqueous mobile phase maintained at a pre-selected temperature less than about 50° C. during analysis of double-stranded nucleic acids by temperature-compression denaturing high performance liquid chromatography, said mobile phase comprising:

• a nitrogen-containing additive, wherein said nitrogen-containing additive comprises a compound according to the formula:
• 9
• wherein:
• R1, R2, and R3, may be the same or different and are independently selected from the group consisting of hydrogen, methyl, ethyl, hydroxyethyl, and propyl, with the proviso that no more than two of R1, R2, and R3 are hydrogen;
• and
• X is a moiety selected from the group consisting of:
• radicals of the formulas
• ═O;
• →O —CH3;
• —CH2CH3;
• and
• 10
• wherein:
• R4 is selected from the group consisting of methyl and hydrogen and, when combined with R1, forms a pyrrolidine ring;
• R5 is selected from the group consisting of —CO2H, —CH2OH, and —SO3H;
• and
• n is an integer of from 0 to 2;
• with the proviso that, when R1 and R4 form a pyrrolidine ring, no more than one of R2 and R3 is hydrogen.

A kit for use in temperature-compression denaturing high performance liquid chromatography, said kit comprising, in separate containers:

• (a) double-stranded DNA corresponding with the nucleic acid identified by SEQ ID NO:
• 1,
• (b) double-stranded DNA corresponding with the nucleic acid identified by SEQ ID NO:
• 2,
• (c) Pho DNA polymerase.

A chromatographic method for separating heteroduplex and homoduplex DNA molecules in a test mixture, said method comprising:

• (a) applying the test mixture to a reverse phase separation medium,
• (b) eluting the medium of step (a) with a mobile phase comprising betaine, 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 heteroduplexes from said homoduplexes,
• wherein said eluting is effected at a pre-selected concentration of betaine and a at a pre-selected temperature,
• wherein said betaine is present at a concentration at which first heteroduplex molecules from a first mixture and second heteroduplex molecules from a second mixture can be observed at said pre-selected temperature, and wherein said first heteroduplex molecules have different a value of T(hsst) than said second heteroduplex molecules.

A method for selecting a concentration of betaine to be used in mobile phase during temperature-compression denaturing high performance liquid chromatography, the method comprising:

• (a) applying a first mixture comprising first heteroduplex molecules and first homoduplex molecules to a reverse phase separation medium,
• (b) eluting the medium of step (a) with a mobile phase comprising betaine, wherein said eluting is carried out under conditions effective to at least partially denature said first heteroduplexes and wherein the eluting results in separation of said first heteroduplexes from said first homoduplexes, wherein said eluting in step (a) is effected at a pre-selected temperature,
• (c) applying a second mixture comprising second heteroduplex molecules and second homoduplex molecules to said reverse phase separation medium,
• (d) eluting the medium of step (c) with a mobile phase comprising betaine, wherein said eluting is carried out under conditions effective to at least partially denature said second heteroduplexes and wherein the eluting results in separation of said second heteroduplexes from said second homoduplexes,
• wherein said eluting is effected at said pre-selected temperature,
• (e) selecting a betaine concentration in the range of about 2M to about 5M at which said first heteroduplexes can be detected in step (c) and wherein said second heteroduplexes can be detected in step (d),
• wherein said first mixture comprises a hybridization product of the double stranded sequence variant 168A and sequence variant 168G, wherein the 168A variant corresponds to the nucleic acid identified by SEQ ID NO:
• 1,
• wherein said second mixture comprises a hybridization product of the double stranded sequence variant 46C and sequence variant 46G, wherein the 46C variant corresponds to the nucleic acid identified by SEQ ID NO:
• 2.