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.

Abstract: The disclosure describes an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein. The separation media has nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.

Abstract: Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.

Abstract: An improved separation column and method for separating a mixture of double stranded DNA fragments by Matched Ion Polynucleotide Chromatography. The cylindrical column has an ID greater than about 5 mm and contains polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA. The improved column provides enhanced separation of DNA fragments with sizes ranging from about 100 to 20,000 base pairs. The column also provides enhanced separation of heteroduplex and homoduplex DNA molecules in a mutation detection procedure in which the chromatography is performed under conditions effecting partial denaturation of DNA at a site of mismatched base pairs.

Abstract: The present invention is a multiple channel electrophoresis system which includes a multiple laser diode illumination source. The multiple laser diode illumination source has a plurality of individual laser diodes, each of which is situated so as to provide independent fluorescence inducing illumination to one of the electrophoresis channels in the multiple channel electrophoresis system. The channels in the multiple channel electrophoresis system can be of many embodiments. And use of the present invention system in a multiple angle light scattering mode is also described.

Abstract: Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surface of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from multivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched ion Polynucleotide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.

Abstract: A liquid chromatography apparatus with stationary and mobile phase temperature controls suitable for polynucleotide separations by MIPC and DMIPC processes. The apparatus includes heater means with a temperature control system; a matched ion polynucleotide chromatography separation column having an inlet end; a coil of capillary tubing having an inlet end and an outlet end. The outlet end of the capillary tubing is connected with the inlet end of the separation column. The inlet end of the capillary tubing comprising means for receiving process liquid, the tubing having a length of from 6 to 400 cm having a linear tubing length of heating means. The separation column and the coil of capillary tubing are enclosed in the heater means. The capillary tubing preferably is PEEK or titanium. The heater means can be an air batch oven. Preferably, it is a heat-conducting block having a first heat transfer surface, a separation column receptacle, and a capillary coil receptacle.

Abstract: A method and apparatus for representing double stranded nucleic acid fragments which have been separated by a chromatographic process as an array of bands which can be accurately quantified, optimized and stored. Using, for example, a Matched Ion Polynucleotide Chromatography (MIPC) process, an analog output from a UV detector is digitized and input to a computer. The digitized signal is converted to a linear array of bands which may be displayed on a video display terminal. The intensity and/or color of a band may correlate to the amount of double stranded nucleic acid in the respective fraction or the respective double stranded nucleic acid fragment above a user selected threshold level at a corresponding point in the digitized signal. The calculated base pair length, concentration, and retention time of each band in the array of bands may be displayed in alphanumeric form.

Abstract: Mixtures of dsDNA fragments are separated by Matched Ion Polynucleotide Chromatography (MIPC) using an isocratic mobile phase to elute polynucleic acid from an MIPC column. The use of isocratic elution conditions provides a marked improvement in the separation of dsDNA fragments compared to gradient elution conditions. Isocratic elution can also be used to effect an improved separation of heteroduplex and homoduplex mixtures when the chromatography is performed under partially denaturing conditions. In addition, dsDNA fragments are bound to the stationary phase under isocratic conditions until a solvent concentration is reached which releases fragments of a particular base pair length range. This separation process is different from the equilibrium partitioning process observed under gradient elution conditions.

Abstract: Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surfaces of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from mutivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.

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.

Abstract: A method for removing a target DNA fragment having a predetermined base-pair length from a mixture of DNA fragments comprises the following steps. A mixture of DNA fragments which may contain the target DNA fragments is applied to a separation column containing media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent. The target DNA fragments are separated from the media by contacting it with a second solvent solution containing a counterion and a concentration of driving solvent in cosolvent which has been predetermined to remove DNA fragments having the target DNA fragment base pair length from the media. The target DNA fragments can be collected and optionally amplified. When the method is being applied to collect a putative fragment, if present, no DNA fragments having the base pair length of the target DNA could be present in the mixture.

Abstract: Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.

Abstract: Improved liquid chromatography systems having components made of titanium, stainless steel, or organic polymeric material are useful in the separation of polynucleotide fragments, particularly large fragments of double-stranded polynucleotides, by Matched Ion Polynucleotide Chromatography (MIPC). The titanium, stainless steel, or polymeric components are treated so that they do not release multivalent cations into aqueous solutions flowing through the chromatography system. Alternatively, or in addition to utilizing materials made of the components listed above, a multivalent cation capture resin placed upstream of the separation column can be employed to remove multivalent ions from the system. The multivalent cation capture resin can be contained in a guard disk, a guard column, or a guard cartridge.

Abstract: A method for detecting a putative mutant DNA in a sample of DNA includes the steps of amplifying the sample of DNA using PCR; hybridizing the amplified sample to form a mixture of homoduplexes and heteroduplexes; separating the mixture into fractions by Denaturing Matched Ion Polynucleotide Chromatography; and blind collecting the eluted fractions at a retention time corresponding to the retention time of the heteroduplex. The DNA in the blind collected fractions can be PCR amplified to obtain an increased amount of heteroduplex relative to homoduplex. The method is useful for determining the remission status of a patient in which the tissue-derived DNA sample contains a large background of wild type or where the putative mutant DNA is below the limit of detection.

Abstract: Covalently bound non-polar tags are used to increase the retention times of double stranded polynucleotides on Matched Ion Polynucleotide Chromatography (MIPC) columns. In doing so, separations of DNA mixture components is improved. Additionally, when the non-polar tags are fluorophores, detection limits are also greatly reduced. Strategically tagged primers are used in conduction with PCR to produce DNA fragments having specifically tagged strands. This improves mutation detection by MIPC in several ways. Separations are improved, detection sensitivity is enhanced, and non-stoichiometric addition of wild type DNA prior to hybridization is now possible since only tagged fragments will be observed with a fluorescence detector. Non-polar tags are also used as a novel alternative to G-C clamping during MIPC under partially denaturing conditions. Reversible DNA binding dyes, such as DNA intercalator dyes and DNA groove binding dyes, are used to reduce the detection limit of polynucleotides separated by MIPC.

Abstract: The present invention provides a method for calibrating a MIPC column wherein the calibration relates to the determination of the organic solvent component in the mobile phase required to elute dsDNA fragments of different base pair lengths at specific retention times. Since a MIPC column affords highly reproducible separations, once calibrated, the base pair length of unknown dsDNA fragments can be determined by comparing their retention times to those obtained on a standard calibration chromatogram. The standard calibration chromatogram is obtained by chromatographing a standard dsDNA ladder containing fragments of known base pair length. In addition, a method is provided to determine the presence of nicks in dsDNA using MIPC under fully denaturing conditions, e.g., 80° C. In one embodiment, this method is applied to the detection of mutations in dsDNA.

Abstract: A batch process for obtaining polynucleotide fragments, such as dsDNA, having a selected size from a mixture of polynucleotide fragments including the steps of a) applying a solution of the mixture of polynucleotide fragments and a counterion agent to a binding medium having a hydrophobic surface; b) contacting the binding medium with a first stripping solvent and counterion agent, the first stripping solvent having a concentration of organic component sufficient to release from the binding medium all polynucleotide fragments having a size smaller than the selected size, and removing the first stripping solvent from the binding medium; and c) contacting the binding medium with a second stripping solvent having a concentration of organic component sufficient to release from the binding medium the polynucleotide fragments having the selected size, and removing the second stripping solvent from the binding medium. The binding medium can be organic polymer or inorganic particle beads.

Abstract: Improved liquid chromatography systems having components made of titanium, stainless steel, or organic polymeric material are useful in the separation of polynucleotide fragments, particularly large fragments of double-stranded polynucleotides, by Matched Ion Polynucleotide Chromatography (MIPC). The titanium, stainless steel, or polymeric components are treated so that they do not release multivalent cations into aqueous solutions flowing through the chromatography system. Alternatively, or in addition to utilizing materials made of the components listed above, a multivalent cation capture resin placed upstream of the separation column can be employed to remove multivalent ions from the system. The multivalent cation capture resin can be contained in a guard disk, a guard column, or a guard cartridge.

Abstract: The invention recognizes the deleterious effects of trace, and even undetectable amounts of multivalent cations on the separation of mixtures of polynucleotides, especially double stranded polynucleotides, and provides an improved method for separating such mixtures on wide pore, non-polar separation media by eliminating multivalent cations from the all aspects of the separation process. This is accomplished by using components in the separation process which are materials which do not release metal cations. In addition, the use of cation capture resins and other methods to remove residual traces of multivalent cations from eluting solvents, sample solutions, separation media, and system components is described. It is also important to remove any traces or organic contaminants from solvents solutions and system parts.