Mutation detection method
AssigneeTransgenomic, Inc.
InventorsDouglas T. Gjerde, Paul D. Taylor, Robert M. Haefele, Christopher P. Hanna
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Abstract
The present invention is directed to improved methods for detection of mutations in DNA using Denaturing Matched Ion Polynucleotide Chromatography (DMIPC). The invention includes the following aspects: analysis of PCR amplification products to identify factors that affect PCR replication fidelity; design of PCR primers; selection of an optimal temperature for performing DMIPC; selection of the mobile phase composition for gradient elution; methods for column preparation and maintenance; and methods for preparing polynucleotide samples prior to chromatographic analysis.
Claims79
In an improved method for separating a sample mixture of polynucleotides by Matched Ion Polynucleotide Chromatography wherein the concentration of polynucleotides in the sample mixture is below a determined threshold concentration, the improvement comprising accumulating the sample mixture of polynucleotides by applying the sample mixture to the column.
A method for preparing a double stranded DNA fragment for mutation detection by Denaturing Matched Ion Polynucleotide Chromatography, the double stranded DNA fragment corresponding to a wild type double stranded DNA fragment having a known nucleotide sequence, comprising the steps of:
- (a) analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences of nucleotides having a melting point range of less than 15 degrees C., each sample sequence having a first end and a second end opposite thereto;
- and
- (b) amplifying one of said sample sequences by PCR using a set of primers which flank the first and second ends of said one of said sample sequences.
A method for mutation detection of a double stranded DNA fragment by Denaturing Matched Ion Polynucleotide Chromatography comprising the steps of
- (a) analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences of nucleotides having a melting point range of less than 15 degrees C., each sample sequence having a first end and a second end opposite thereto;
- (b) amplifying one of said sample sequences by PCR using a set of primers which flank the first and second ends of said one of said sample sequences;
- (c) analyzing the amplified sample by Denaturing Matched Ion Polynucleotide Chromatography.
A method for evaluating a PCR process to determine if it induces mutations, the method comprising the steps of
- a) amplifying a polynucleotide by performing a plurality of PCR process cycles to yield a PCR amplification product;
- b) analyzing the PCR amplification product by Matched Ion Polynucleotide Chromatography to yield a PCR amplification product profile, including a profile of any mutations produced by PCR produced mutation.
A method for identifying deviations of a PCR process from a predetermined reference profile, the method comprising
- a) amplifying a polynucleotide by performing a plurality of PCR process cycles to yield a PCR amplification product;
- b) analyzing the PCR amplification product by Matched Ion Polynucleotide Chromatography to yield a PCR amplification product profile, including a profile of any PCR induced mutations.
A method for reducing PCR induced mutation in a PCR amplification process, the method comprising
- a) amplifying a polynucleotide by performing a plurality of PCR amplification process cycles to yield a first PCR amplification product;
- b) analyzing the first PCR amplification product by Matched Ion Polynucleotide Chromatography to yield a PCR amplification product profile;
- c) comparing the PCR amplification product profile against a reference profile to determine the presence of PCR induced mutations;
- d) amplifying a polynucleotide by performing a plurality of PCR amplification process cycles with an adjustment of one or more process variables to form a second PCR amplification product with reduced PCR induced mutations.
A method for detecting DNA genetic mutations comprising the steps of
- 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 Matched Ion Polynucleotide Chromatography at the heteromutant site separation temperature to identify the presence of any heteromutant site separated components therein.
A method for detecting DNA genetic mutations comprising the steps of
- a) a calculation step for obtaining a calculated heteromutant site separation temperature;
- b) a prediction step for obtaining a predicted heteromutant site separation temperature;
- c) heating a mixture of a sample double stranded DNA segment and a corresponding wild type double stranded DNA segment to the predicted heteromutant site separation temperature;
- d) analyzing the product of step (c) 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 chromatographic method for separating a mixture of heteroduplex and homoduplex DNA molecules including a first eluting DNA molecule and a last eluting DNA molecule, under conditions which selectively denature a mutation site present in the heteroduplex DNA molecule, comprising the steps of:
- (a) applying the mixture to a Matched Ion Polynucleotide Chromatographic column,
- (b) eluting the molecules of said mixture using a mobile phase comprising a counterion agent and a pre-selected fragment bracketing range of organic solvent concentration, said range comprising an initial concentration and a final concentration of organic solvent, said initial concentration containing an organic solvent concentration up to an amount required to elute the first eluting DNA molecule in the mixture, and said final concentration containing an organic solvent concentration sufficient to elute the last eluting DNA molecule in the mixture.
A method for treating a matched ion polynucleotide chromatography column in order to improve the resolution of double stranded DNA fragments separated on said column comprising flowing a solution containing a multivalent cation binding agent through said column, wherein said solution has a temperature of about 50 to 90° C.
A method for storing a Matched Ion Polynucleotide Chromatography column in order to improve the resolution of double stranded DNA fragments separated on said column comprising flowing a solution containing a multivalent cation binding agent through said column prior to storing said column.
A method for separating a mixture of double stranded polynucleotides, comprising flowing a mixture of polynucleotides having up to 1500 base pairs through a separation column containing polymer beads having an average diameter of 0.5 to 100 microns, wherein said beads are characterized by having a Mutation Separation Factor of at least 0.01, and separating said mixture of polynucleotides.
A method for preparing a double stranded DNA fragment for mutation detection by Denaturing Matched Ion Polynucleotide Chromatography, the double stranded DNA fragment corresponding to a wild type double stranded DNA fragment having a known nucleotide sequence, comprising the steps of:
- (a) analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences of nucleotides having a high melting domain and a low melting domain in which a mutation site is located;
- and
- (b) amplifying one of said sample sequences by PCR using a set of primers which flank the first and second ends of said sample sequences.
A method for preparing a double stranded DNA fragment for mutation detection by Denaturing Matched Ion Polynucleotide Chromatography, the double stranded DNA fragment corresponding to a wild type double stranded DNA fragment having a known nucleotide sequence, comprising the steps of:
- (a) analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences of nucleotides wherein the mutation site is within twenty-five percent of the total number of base pairs from an end of the fragment;
- and
- (b) amplifying one of said sample sequences by PCR using a set of primers which flank the first and second ends of said sample sequences.
Dependencies are inferred from the claim text.