Transgenomic, Inc.

Other

Paul D Taylor

Douglas T Gjerde

Robert M Haefele

Christopher P Hanna

Gary Floyd Gerard

David P Hornby

John E Sutton

Kimberly A Lamb

Reyes Candau

Benjamin L Legendre, Jr

Robert M Hae Fele

Alexander I Kuklin

Satyajit Kar

Alezander I Kuklin

Laura Bunnel

Liem T Nguyen

The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3' side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3&#x2032;-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule

The invention relates to methods for identifying the sequence of one or more variant nucleotides in nucleic acid molecules. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3' side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a Double-Stranded Linker with a degenerate 3&#x2032;-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

In an extensive Matched Ion Polynucleotide Chromatography (MIPC) system and method, and the computer programs or software associated therewith, the system provides automated options for sample selection, mobile phase gradient selection and control, column and mobile phase temperature control, and fragment collection for a wide variety of MIPC separation processes. MIPC separation processes can be applied to effect size-based separation of DNA fragments, mutation detection, DNA fragment purification, PCR process monitoring and other novel processes. This invention is directed to the system and software which automates many of these procedures, facilitating use of the system to achieve complex separation methods. In one embodiment of the invention, a user specifies a size range of double stranded DNA fragment(s) in a mixture, the software calculates a solvent gradient to elute the fragment(s), and the system performs the chromatographic separation using the calculated gradient. In an embodiment useful in DNA mutation detection, a user specifies the base sequence of a wild type DNA molecule, the software calculates a temperature for partially denaturing heteroduplex and homoduplex molecules of the DNA in a mixture, the software calculates a solvent gradient to elute the fragments, and the system performs the chromatographic separation using the calculated gradient and temperature.

System and method for automated matched ion polynucleotide chromatography

In one aspect, the invention provides a method for separating a mixture of polynucleotides, such as DNA or RNA, including (a) applying the mixture to a polymeric separation medium having non-polar surfaces, wherein the surfaces are characterized by being substantially free from multivalent cations, such as metal ions, which are free to interfere with polynucleotide separation, and (b) eluting the mixture with a mobile phase containing organic solvent and counter ion agent. In the separation of single-stranded polynucleotides, improved separation is obtained at a temperature effective to fully denature secondary structure within the polynucleotides.

Liquid chromatographic separation of polynucleotides

Methods, compositions, and kits for separating heteroduplex and homoduplex DNA molecules in a test mixture by temperature-compression denaturing high performance liquid chromatography (tcDHPLC). The method includes use of nitrogen-containing additives in the mobile phase that allow detection of diverse heteroduplex molecules to be performed at the same pre-selected temperature. An example of a preferred additive is betaine. Standard mixtures of DNA fragments, such as mutation standards containing known heteroduplex and homoduplex molecules, can be used to select the concentration of additive and temperature. Compositions and kits including the mobile phase, mutation standards, PCR primers, separation media, and DNA polymerase are also provided.

Methods and compositions for mutation analysis of polynucleotides by liquid chromatography

The present invention describes a method for separating or partially separating heteroduplex and homoduplex DNA molecules in a mixture. In the method, the mixture is applied to an anion-exchange chromatography medium. The heteroduplex and homoduplex molecules are eluted with a mobile phase containing an eluting salt, including an anion and a cation, a buffer, and preferably including an organic solvent. The eluting is carried out under conditions effective to at least partially denature the heteroduplexes (e.g., thermal or chemical denaturing) resulting in the separation of the heteroduplexes from the homoduplexes. The method has many applications including, but not limited to, detecting mutations and comparative DNA sequencing.

Detection of nucleic acid heteroduplex molecules by anion-exchange chromatography

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.

Modifying double stranded DNA to enhance separations by matched ion polynucleotide chromatography

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. The mixture of polynucleotides can be the product of a PCR amplification. The binding medium can be contained within a column, a web or a container.

Process for separation of polynucleotide fragments

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.

Method and system for RNA analysis by matched ion polynucleotide chromatography

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.

Non-polar media for polynucleotide separations

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.

Polynucleotide separations on polymeric separation media

An apparatus for effecting base pair length separations of DNA fragments by matched ion paired chromatography comprising a separation column containing separation media having non-polar DNA separation surfaces, separation solution supply means, and a separation solution conduit communicating with the separation column and the separation solution supply means, and a cleaning solution valve means positioned in the separation solution conduit for injecting cleaning solution into the separation solution conduit. A process for cleaning the non-polar DNA separation surfaces in the apparatus comprising interrupting the flow of separation solvent with a block of cleaning solution injected into the flow of separation solution passing to the column, the cleaning solution containing agent which removes accumulated residues from the non-polar surface. The cleaning solution can have an alkaline pH and contain a chelating agent such as EDTA.

MIPC column cleaning system and process

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.

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Gilroy, CA, United States of America

Paul D Taylor