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WO2001092510A1 - Appareil et procede permettant la separation et la purification de polynucleotides - Google Patents

Appareil et procede permettant la separation et la purification de polynucleotides Download PDF

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Publication number
WO2001092510A1
WO2001092510A1 PCT/US2000/014956 US0014956W WO0192510A1 WO 2001092510 A1 WO2001092510 A1 WO 2001092510A1 US 0014956 W US0014956 W US 0014956W WO 0192510 A1 WO0192510 A1 WO 0192510A1
Authority
WO
WIPO (PCT)
Prior art keywords
separation
dna
fragments
dna fragments
heteroduplex
Prior art date
Application number
PCT/US2000/014956
Other languages
English (en)
Inventor
Douglas T. Gjerde
Christopher P. Hanna
Paul D. Taylor
Benjamin L. Legendre, Jr.
Robert M. Haefele
Original Assignee
Transgenomic, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Transgenomic, Inc. filed Critical Transgenomic, Inc.
Priority to CA002396667A priority Critical patent/CA2396667A1/fr
Priority to JP2002500702A priority patent/JP2003534798A/ja
Priority to PCT/US2000/014956 priority patent/WO2001092510A1/fr
Priority to AU54517/00A priority patent/AU5451700A/en
Publication of WO2001092510A1 publication Critical patent/WO2001092510A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/287—Non-polar phases; Reversed phases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/161—Temperature conditioning
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/366—Ion-pair, e.g. ion-pair reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • Fig. 14 is a chromatogram of the separated 102 bp fragment obtained in the procedure of Example 1.
  • Fig. 15 is a chromatogram of the separated 174 bp fragment obtained in the procedure of Example 1.
  • the methods of this invention for isolating targeted DNA fragments having a predetermined base-pair length from a mixture of DNA fragments comprise the following steps.
  • a mixture of DNA fragments is applied to a separation column containing separation media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solution containing counterion and a DNA binding concentration of driving solvent.
  • the targeted DNA fragments are then removed from the media by contacting the media with a second solution containing counterion and a concentration of driving solvent which has been predetermined to remove DNA fragments having the targeted fragments from the separation media into a distinct segment of eluant.
  • the present invention relates, therefore, to the isolation of DNA fragments having a predetermined base pair (bp) length from a mixture of fragments.
  • the present invention provides a method for enhancing the separation dsDNA fragments on an MIPC column by using an isocratic mobile phase to elute fragments from the column.
  • Separations of dsDNA fragments by MIPC under isocratic elution conditions are extremely sensitive to solvent composition. Small changes in the composition of solvent mixtures contained in solvent reservoirs can occur as a result of evaporation. Such changes can result in differences in the retention times of fragments from identical samples.
  • DMIPC DMIPC. If the original sample contained a mutation, the concentration of heteroduplex or its concentration relative to wild type, will now be sufficient to detect. The DMIPC chromatogram will, therefore, show a peak having the retention time of the standard heteroduplex. In this event it can be concluded unambiguously that a mutation was present in the original sample.
  • Non-limiting examples of counterion agents include octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylamrhonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethy
  • the separation vial components142 and 146 of Fig. 7 and the plates 200 and 220 in Figs. 8-11 are made of a material which does not interfere with the separation process such as polystyrene, polypropylene, or polycarbonate.
  • the upper plate 206 and housing 210 can be made of any materials having the requisite strength such as a rigid organic polymer, aluminum, stainless steel or the like.
  • the vacuum chamber walls are preferably coated with Teflon film.
  • the vacuum conduit and valve can also be made of Teflon coated aluminum or the like.
  • the vacuum apparatus and plates are reassembled, and a second aqueous solution containing counterion and a second higher concentration of driving solvent is prepared and placed in the chambers 202.
  • the driving solvent concentration is calculated to be the amount which will remove all lower molecular weight material from the separation media.
  • the precise concentration of driving solvent is determined from a standard table or curve, or it can be calculated, using values determined in a calibration of the media made using a double-stranded DNA (dsDNA) ladder or standardized enzyme digest.
  • primer extension products Once the primer extension products are selectively released, they can be sized directly on the DNA Chromatography matrix ( Hoogendoorn, B., et al "Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography", Human Genetics, 1999, Vol. 104, No. 1 , pp. 89-93. If the purified primer extension products are collected, they can also be sized by other means such as gel electrophoresis or mass spectrometry. When the purification procedures noted above are applied, it is possible to detect the purified products either by UV absorbance, or by fluorescent intensity.
  • the ability to size the fragments prior to mass spectrometric analysis is of great utility, particulariy when trying to perform more than one primer extension at a time (i.e. multiplexing).
  • MALDI- TOF mass spectrometry
  • a target product requiring determination is a 20-mer
  • mass spectrometry it is theoretically possible to have masses from 18-mers to 22-mers overlapping with the theoretical masses of the 20-mer product. Therefore, separating the primer extension products on the basis of size prior to their analysis by mass spectrometry effects a real-time deconvolution of the data, thus making interpretation more simple.
  • the probes are then selectively eluted from the DNA Chromatography matrix.
  • the "mass tags” are cleaved from the probes and then conducted to the mass spectrometer for detection (and hence detection of the probing event). This can further simplify the detection of "mass tags” by providing another dimension along which selectivity can be provided, i.e., the dimension of probe length.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Plant Pathology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Electrochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un procédé permettant d'éliminer un fragment d'ADN cible ayant une longueur de paires de bases prédéterminée, à partir d'un mélange de fragments d'ADN, ledit procédé comprenant les étapes suivantes: un mélange contenant des fragments d'ADN, qui peut contenir les fragments d'ADN cible, est appliqué à une colonne de séparation contenant un milieu ayant une surface non poreuse, non polaire, le mélange de fragments d'ADN se trouvant dans un premier mélange de solvants contenant un ion antagoniste et une concentration de liaison d'ADN d'un solvant d'entraînement dans un cosolvant; les fragments d'ADN cible sont séparés du milieu grâce à leur mise en contact avec une deuxième solution de solvants contenant un ion antagoniste et une concentration de solvant d'entraînement dans un cosolvant qui a été préparé pour permettre d'éliminer du milieu les fragments d'ADN ayant la longueur de paires de bases de fragments d'ADN cible. Les fragments d'ADN cible peuvent être recueillis et éventuellement amplifiés. Lorsque le procédé est appliqué pour recueillir un fragment putatif, si celui-ci est présent, aucun fragment d'ADN ayant la longueur de paires de bases de l'ADN cible ne peut être présent dans le mélange. De façon alternative, des fragments d'ADN ayant la longueur de paires de bases de l'ADN cible sont présents dans le mélange. Cette invention concerne également un dispositif à pression ambiante ou à basse pression, servant à séparer des fragments polynucléotidiques d'un mélange de fragments polynucléotidiques, ledit dispositif comprenant un tube présentant un compartiment supérieur d'entrée de solution, un compartiment inférieur de réception d'éluant, et une unité fixe du milieu de séparation qui s'y trouve. Le milieu de séparation présente des surfaces non polaires qui sont dépourvues de cations plurivalents qui réagiraient sinon avec l'ion antagoniste pour former une couche polaire insoluble à la surface du milieu de séparation.
PCT/US2000/014956 2000-05-26 2000-05-26 Appareil et procede permettant la separation et la purification de polynucleotides WO2001092510A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002396667A CA2396667A1 (fr) 2000-05-26 2000-05-26 Appareil et procede permettant la separation et la purification de polynucleotides
JP2002500702A JP2003534798A (ja) 2000-05-26 2000-05-26 ポリヌクレオチドを分離しそして精製するための装置及び方法
PCT/US2000/014956 WO2001092510A1 (fr) 2000-05-26 2000-05-26 Appareil et procede permettant la separation et la purification de polynucleotides
AU54517/00A AU5451700A (en) 2000-05-26 2000-05-26 Apparatus and method for separating and purifying polynucleotides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2000/014956 WO2001092510A1 (fr) 2000-05-26 2000-05-26 Appareil et procede permettant la separation et la purification de polynucleotides

Publications (1)

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WO2001092510A1 true WO2001092510A1 (fr) 2001-12-06

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AU (1) AU5451700A (fr)
CA (1) CA2396667A1 (fr)
WO (1) WO2001092510A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073732A3 (fr) * 2004-01-23 2006-01-19 Amgen Inc Procede lc/ms d'analyse de proteines de poids moleculaire eleve

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020162485A (ja) * 2019-03-29 2020-10-08 積水化学工業株式会社 核酸の回収方法、核酸増幅方法、及びマイクロ流体デバイス

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994011305A1 (fr) * 1992-11-18 1994-05-26 Sarasep, Incorporated Separation de fragments d'acide nucleique
WO1998040395A1 (fr) * 1997-03-14 1998-09-17 Transgenomic, Inc. Affichage de separations polyonucleotidiques sous la forme d'un arrangement de bandes
WO1998048914A1 (fr) * 1997-04-25 1998-11-05 Transgenomic, Inc. Milieu chromatographique liquide ameliore utilise dans la separation de polynucleotides
WO1998048913A1 (fr) * 1997-04-25 1998-11-05 Transgenomic, Inc. Separations de polynucleotides sur des billes de polymeres non poreuses
WO1998056797A1 (fr) * 1997-06-10 1998-12-17 Transgenomic, Inc. Systeme et procede permettant de realiser des separations de polynucleotides grace a la chromatographie en phase liquide
WO1999019514A1 (fr) * 1997-10-09 1999-04-22 Transgenomic, Inc. Modification d'adn double brin pour ameliorer les separations par chromatographie de polynucleotides a ions apparies

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994011305A1 (fr) * 1992-11-18 1994-05-26 Sarasep, Incorporated Separation de fragments d'acide nucleique
WO1998040395A1 (fr) * 1997-03-14 1998-09-17 Transgenomic, Inc. Affichage de separations polyonucleotidiques sous la forme d'un arrangement de bandes
WO1998048914A1 (fr) * 1997-04-25 1998-11-05 Transgenomic, Inc. Milieu chromatographique liquide ameliore utilise dans la separation de polynucleotides
WO1998048913A1 (fr) * 1997-04-25 1998-11-05 Transgenomic, Inc. Separations de polynucleotides sur des billes de polymeres non poreuses
WO1998056797A1 (fr) * 1997-06-10 1998-12-17 Transgenomic, Inc. Systeme et procede permettant de realiser des separations de polynucleotides grace a la chromatographie en phase liquide
WO1999019514A1 (fr) * 1997-10-09 1999-04-22 Transgenomic, Inc. Modification d'adn double brin pour ameliorer les separations par chromatographie de polynucleotides a ions apparies

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Title
GELFI C ET AL: "DETECTION OF POINT MUTATIONS BY CAPILLARY ELECTROPHORESIS IN LIQUID POLYMERS IN TEMPORAL THERMAL GRADIENTS", ELECTROPHORESIS,DE,WEINHEIM, vol. 15, no. 12, 1 December 1994 (1994-12-01), pages 1506 - 1511, XP000671721 *
HUBER C G ET AL: "RAPID AND ACCURATE SIZING OF DNA FRAGMENTS BY ION-PAIR CHROMATOGRAPHY ON ALKYLATED NONPOROUS POLY(STYRENE- DIVINYLBENZENE) PARTICLES", ANALYTICAL CHEMISTRY,US,AMERICAN CHEMICAL SOCIETY. COLUMBUS, vol. 67, 1 February 1995 (1995-02-01), pages 578 - 585, XP002911660, ISSN: 0003-2700 *
KUKLIN A ET AL: "A novel technique for rapid automated genotyping of DNA polymorphisms in the mouse.", MOLECULAR AND CELLULAR PROBES, vol. 13, no. 3, June 1999 (1999-06-01), pages 239 - 242, XP002159014, ISSN: 0890-8508 *
MYERS R M ET AL: "MODIFICATION OF THE MELTING PROPERTIES OF DUPLEX DNA BY ATTACHMENT OF A GC-RICH DNA SEQUENCE AS DETERMINED BY DENATURING GRADIENT GEL ELECTROPHORESIS", NUCLEIC ACIDS RESEARCH,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 13, no. 9, 1985, pages 3111 - 3129, XP002911668, ISSN: 0305-1048 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073732A3 (fr) * 2004-01-23 2006-01-19 Amgen Inc Procede lc/ms d'analyse de proteines de poids moleculaire eleve
US7329353B2 (en) 2004-01-23 2008-02-12 Amgen Inc. LC/MS method of analyzing high molecular weight proteins

Also Published As

Publication number Publication date
JP2003534798A (ja) 2003-11-25
AU5451700A (en) 2001-12-11
CA2396667A1 (fr) 2001-12-06

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