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 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: The instant invention provides a non-HPLC chromatographic method for purifying a target polynucleotide comprising the steps of: applying the target polynucleotide to a separation medium having a non-polar separation surface in the presence of a counterion agent, whereby the polynucleotide is bound to the separation medium; eluting the target polynucleotide from the separation medium by passing through the separation medium an elution solution containing a concentration of organic solvent sufficient to elute the target polynucleotide from the separation medium; and collecting the eluted target polynucleotide. The separation medium can be supported in any of a variety of containers, non-limiting preferred examples of which include spin columns and vacuum trays. The invention is particularly useful for the separation of RNA and single and double stranded DNA.

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: 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: 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: 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 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: 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: The instant invention provides a method for stabilizing an RNA molecule against degradation comprising applying a solution to a separation medium having a non-polar separation surface in the presence of a counterion agent, wherein the solution comprises the RNA molecule and an agent capable of catalyzing the degradation of RNA; eluting the RNA molecule from the separation medium by passing through the separation medium a mobile phase containing a concentration of organic solvent sufficient to elute the RNA molecule from the separation medium, where the elution is conducted under conditions that result in a substantial separation of the RNA molecule from the agent capable of catalyzing the degradation of RNA; and collecting an eluant fraction containing the RNA molecule that is substantially free of the agent capable of catalyzing the degradation of RNA. In a preferred embodiment the method is performed under conditions that are substantially free of multivalent cations.

Abstract: A Matched Ion Polynucleotide Chromatography method and system for size-based segregation of a mixture of RNA molecules. The method includes applying the mixture to a polymeric separation medium having non-polar surfaces and eluting the RNA molecules with a mobile phase which includes counterion reagent and an organic component. The preferred surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation. The elution is preferably performed at a temperature sufficient to denature the RNA. The method can be used in segregating RNA molecules having lengths in the range of about 100 to 20,000 nucleotides. Improved segregation is obtained using a chromatography column having an ID greater than about 5 mm. Examples of separation media include beads and monolithic columns.

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: 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 Polynucleolide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.

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: 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: 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: 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.