EP1339876A2 - Purification de reactions de sequen age d'adn au moyen de particules magnetiques de silice - Google Patents

Purification de reactions de sequen age d'adn au moyen de particules magnetiques de silice

Info

Publication number
EP1339876A2
EP1339876A2 EP01998656A EP01998656A EP1339876A2 EP 1339876 A2 EP1339876 A2 EP 1339876A2 EP 01998656 A EP01998656 A EP 01998656A EP 01998656 A EP01998656 A EP 01998656A EP 1339876 A2 EP1339876 A2 EP 1339876A2
Authority
EP
European Patent Office
Prior art keywords
complex
dna
magnetic particles
silica magnetic
solution
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP01998656A
Other languages
German (de)
English (en)
Inventor
Michael P. Bjerke
Paul E. Otto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Promega Corp
Original Assignee
Promega Corp
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 Promega Corp filed Critical Promega Corp
Publication of EP1339876A2 publication Critical patent/EP1339876A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • This invention relates generally to methods of purifying DNA extension products from a DNA sequencing reaction containing unincorporated dideoxynucleotides, primers, salts, and other materials which might adversely affect analysis of the DNA extension products of the sequencing reactions.
  • This invention relates, particularly to the use of silica matrices, particularly silica magnetic particles, in purifying DNA extension products from DNA sequencing reactions, including DNA sequencing reactions wherein dideoxynucleotides labeled with fluorescent dyes or primers labeled with fluorescent dyes are used.
  • a dideoxy-terminated DNA sequencing reaction the DNA template to be sequenced, all four deoxynucleotides (i.e., dATP, dCTP, dGTP, and dTTP) or functional equivalents thereof (e.g., dITP or dUTP), a polymerase, a dideoxy nucleotide, and a primer are all present.
  • a dideoxy-terminated DNA sequencing reaction (a sequencing reaction that utilizes dideoxynucleotides to halt DNA extension) is initiated by an oligonucleotide primer hybridizing to a complementary sequence of a strand of the template DNA.
  • the polymerase catalyzes a polymerization reaction in which deoxy- or dideoxynucleotides complementary to the corresponding nucleotides on the template DNA are added to the 3' end of the primer, and then to the 3' end of the growing DNA extension product.
  • the polymerization reaction for any given DNA extension product terminates when a dideoxynucleotide is added to the 3' end of the growing extension product.
  • the dideoxy-terminated DNA sequencing reaction was first developed, it was designed so that sequencing took place in four different reactions, with a different one of the four dideoxynucleotides in each reaction (i.e. ddATP, ddCTP, ddGTP, and ddTTP).
  • reaction products can be detected by using 5' labeled primers or 3' labeled dideoxynucleotides, labeled with a radioactive probe or with a fluorescent label, or by incorporation of a radiolabeled deoxynucleotide (e.g. 35 SdATP) or by means that do not require the use of labels.
  • a radiolabeled deoxynucleotide e.g. 35 SdATP
  • the products of such sequencing reactions can be detected by separating the resulting fragments of DNA by gel electrophoresis or by capillary electrophoresis. I cases where the DNA extension products of the DNA sequencing reaction are not labeled, these products are preferably analyzed by separating the products by gel electrophoresis and staining the resulting gel with a DNA sensitive stain, such as a silver stain.
  • a DNA sensitive stain such as a silver stain.
  • radioactive labels the DNA extension products can be detected using any suitable means for detection of radioactivity, such as an autoradiogram of an electrophoresis gel.
  • fluorescent dye labels are used, the DNA extension products can be detected by scanning an electrophoresis gel or capillary array with a fluorescent scanner.
  • Dideoxy-terminated DNA sequencing reactions wherein the dideoxynucleotide(s) is labeled tend to have considerably higher background than do reactions wherein the primer is labeled.
  • high background interferes with read accuracy, particularly within the first 100 bases of the primer.
  • High backgrounds particularly interfere with the reading accuracy of automated fluorescent DNA analysis machines, such as fluorescent gel or capillary electrophoresis analyzers, (e.g., the ABI PRISM ® 377 DNA Sequencer, LI-COR ® 4000 or 4200 Sequencer, ALF DNA SequencerTM, or the ABI PRISM ® 3700 DNA Analyzer).
  • Silica based systems have been developed for use in the purification of plasmid DNA from other material in a solution. Such systems include those which employ controlled pore glass, filters embedded with silica particles, silica gel particles, resins comprising silica in the form of diatomaceous earth, glass fibers or mixtures of the above.
  • Each such silica-based solid phase separation system is configured to reversibly bind nucleic acid materials when placed in contact with a medium containing such materials in the presence of chaotropic agents.
  • the silica-based solid phases are designed to remain bound to the nucleic acid material while the solid phase is exposed to an external force such as centrifugation or vacuum filtration to separate the matrix and nucleic acid material bound thereto from the remaining media components.
  • the nucleic acid material is then eluted from the solid phase by exposing the solid phase to an elution solution, such as water or an elution buffer.
  • an elution solution such as water or an elution buffer.
  • silica-based resins designed for use in centrifugation and/or filtration isolation systems e.g., Wizard ® DNA purification systems products from Promega Corporation (Madison, Wisconsin, U.S.A.), or the QiaPrep ® DNA isolation systems from Qiagen Corp. (Chatsworth, California, U.S.A.).
  • Magnetically responsive solid phases such as paramagnetic or superparamagnetic particles, offer an advantage not offered by any of the silica-based solid phases described above. Such particles could be separated from a solution by turning on and off a magnetic force field, by moving a container on to and off of a magnetic separator, or by moving a magnetic separator on to and off of a container. Such activities would be readily adaptable to automation.
  • Magnetically responsive particles have been developed for use in the isolation of nucleic acids by the direct reversible adsorption of nucleic acids to the particles. See, e.g., silica gel-based porous particles designed to reversibly bind directly to DNA, such as MagneSilTM Paramagnetic Particles (Promega), or BioMag ® Paramagnetic Beads (Polysciences, Warrington, PA, U.S.A.). See also Smith et al., U.S. Patent Number 6,027,945. Magnetically responsive glass beads of a controlled pore size have also been developed for the isolation of nucleic acids. See, e.g. Magnetic Porous Glass (MPG) particles from CPG, Inc.
  • MPG Magnetic Porous Glass
  • the present invention provides a method of purifying a DNA extension product from a DNA sequencing reaction prior to analysis, thereby increasing read length and accuracy of sequence analysis.
  • One embodiment of the method comprises: (a) providing DNA sequencing reaction products comprising an unincorporated primer, an unincorporated dideoxynucleotide, and a DNA extension product; (b) combining the DNA sequencing reaction products with silica magnetic particles in an adsorption solution, wherein the DNA extension products selectively adsorb to the particle, thereby forming a complex; and (c) separating the complex from the adsorption solution.
  • the silica magnetic particles used in this embodiment of the invention are preferably macro-porous silica magnetic particles, or more preferably, low porosity silica magnetic particles.
  • Another embodiment of the method of the present invention is a method of purifying a dye terminal labeled DNA sequencing reaction product, according to the steps comprising: (a) providing dideoxy-terminated DNA sequencing reaction products, comprising an unincorporated dideoxynucleotide labeled with fluorescent dye, and a DNA extension product; (b) combining the DNA sequencing reaction product with silica magnetic particles in an adsorption solution, wherein the DNA extension product selectively adsorbs to the particle, thereby forming a complex; and (c) separating the complex from the adsorption solution.
  • kits for purifying a dideoxy DNA sequencing reaction prior to analysis comprising: a container comprising silica magnetic particles, a chaotropic agent, and a buffer having a pH of less than about 7.0, preferably a buffer having a pH of 5 or less.
  • FIG. 1A is a printout of sequencing results of the first 115 bases of pGEM ® -3Zf DNA template (Promega Cat. # P227) from dye labeled primers each with a sequence identified by SEQ ID NO: 1 using an ABI PRISM 377 DNA Sequencer, after adsorption of the sequencing extension products to MagneSilTM particles (a type of macroporous silica magnetic particle) in a 0.08M potassium acetate (“KOAc”) adsorption solution (pH less than 5.0) and wash of the resulting complex in 70% Ethanol, as described in Example 1.
  • pGEM ® -3Zf DNA template Promega Cat. # P2257
  • Figure IB is a printout of sequencing results of the first 120 bases of pGEM ® -3Zf DNA template (Promega Cat. # P227) from dye labeled primers each with a sequence identified by SEQ ID NO: 1 using an ABI PRISM 377 DNA Sequencer, after ethanol precipitation, as described in Example 1.
  • Figure 2A is a printout of sequencing results of the first 115 bases of pGEM ® -3Zf DNA template (Promega Cat. # P227) from a primer represented by SEQ ID NO: 2 obtained using an ABI PRISM 377 DNA Sequencer and ABI PRISM ® BigDyeTM Terminator reaction mixes, after adsorption of the sequencing extension products to MagneSilTM particles in a 0.6M GTC/0.08M KOAc adsorption solution and wash of the resulting complex in 70% Ethanol, as described in Example 2.
  • Figure 2B is a printout of sequencing results of the first 120 bases of pGEM ® -3Zf + DNA template (Promega Cat. # P227) from a primer represented by SEQ ID NO: 2 obtained using an ABI PRISM 377 DNA Sequencer and ABI PRISM ® BigDyeTM Terminator reaction mixes after ethanol precipitation, as described in Example 2.
  • Figure 3 is a printout of sequencing results of the first 115 bases of pGEM ® -3Zf +
  • DNA template (Promega Cat. # P227) from a primer represented by SEQ ID NO: 2 obtained using an ABI PRISM 377 DNA Sequencer and ABI PRISM ® BigDyeTM
  • Terminator reaction mixes, after adsorption of the sequencing extension products to MagneSilTM particles in a 1M GTC/0.08M KOAc adsorption solution and wash of the resulting complex in 90% Ethanol, as described in Example 3.
  • Figure 4 is a printout of sequencing results of the first 115 bases of pGEM ® -3Zf DNA template (Promega Cat. #P227) from a primer represented by SEQ ID NO: 2 obtained using an ABI PRISM 377 DNA Sequencer and ABI PRISM ® BigDyeTM Terminator reaction mixes, after adsorption of the sequencing extension products to low porosity silica magnetic particles in a 0.4M GTC/0.08M KOAc adsorption solution and two washes of the resulting complex in 80% Ethanol, as described in Example 4.
  • Figure 5 A is a printout of sequencing results of the first 115 bases of pGEM ® -3Zf DNA template (Promega Cat. # P227) from a primer represented by SEQ ID NO: 2 obtained using an ABI PRISM 377 DNA Sequencer and ABI PRISM ® BigDyeTM Terminator reaction mixes, after adsorption of the sequencing extension products to low porosity silica magnetic particles in a 4.35M GTC/0.03M sodium citrate, pH 4.0 adsorption solution, and after wash of the resulting complex in 90% Ethanol, as described in Example 5.
  • Figure 5B is a printout of sequencing results of the first 120 bases of pGEM ® -3Zf l" DNA template (Promega Cat. # P227) from a primer represented by SEQ ID NO: 2 obtained using an ABI PRISM 377 DNA Sequencer and ABI PRISM ® BigDyeTM Terminator reaction mixes after ethanol precipitation, as described in Example 5.
  • chaotropic agent refers to salts of particular ions which, when present in a sufficiently high concentration in an aqueous solution, cause proteins present therein to unfold and nucleic acids to lose secondary structure. It is thought that chaotropic ions have these effects because they disrupt hydrogen-bonding networks that exist in liquid water and thereby make denatured proteins and nucleic acids thermodynamically more stable than their correctly folded or structured counterparts.
  • Chaotropic ions include guanidinium, iodide, perchlorate, and trichloroacetate.
  • Chaotropic agents include guanidine hydrochloride, guanidine thiocyanate (which is sometimes referred to as guanidine isothiocyanate), sodium iodide, sodium perchlorate, and sodium trichloroacetate.
  • the term "magnetic” as used to refer to silica magnetic particles includes materials which are paramagnetic or superparamagnetic materials.
  • the term “magnetic”, as used herein, also encompasses temporarily magnetic materials, such as ferrimagnetic or ferrimagnetic materials.
  • the silica magnetic particles used in this invention preferably comprise a superparamagnetic core coated with siliceous oxide, having a hydrous siliceous oxide adsorptive surface (i.e. a surface characterized by the presence of silanol groups).
  • nucleic acid refers to any DNA or RNA molecule or a DNA/RNA hybrid molecule.
  • the term includes plasmid DNA, DNA or RNA fragments, total RNA, mRNA, genomic DNA, and chromosomal DNA.
  • solid phase is used herein in a standard chromatographic sense, to refer to an insoluble, usually rigid, matrix or stationary phase which interacts with a solute, in this case a DNA extension product in a DNA sequencing reaction.
  • solid phase specifically includes stationary phases in liquid chromatography (LC), high pressure liquid chromatography (HPLC), particulate matrices embedded into or bound to filters, and magnetic or non-magnetic porous matrix particles which interact with solutes when added directly to a solute mixture.
  • silica gel refers to chromatography grade silica gel, a substance which is commercially available from a number of different sources. Silica gel is most commonly prepared by acidifying a solution containing silicate, e.g. by acidifying sodium silicate to a pH of less than 11, and then allowing the acidified solution to gel. See, e.g. silica preparation discussion in Kurt-Othmer Encyclopedia of Chemical Technology, Vol. 21, 4th ed., Mary Howe-Grant, ed., John Wiley & Sons, pub., 1997 , p. 1021.
  • silica magnetic particles refers to silica based solid phases which are further comprised of materials which have no magnetic field but which form a magnetic dipole when exposed to a magnetic field, i.e., materials capable of being magnetized in the presence of a magnetic field but which are not themselves magnetic in the absence of such a field.
  • macroporous silica magnetic particles refers to silica magnetic particles with a median particle size of 1 to 10 ⁇ m and a surface area, as measured by nitrogen BET method, of at least about 10 m 2 /g of particle mass.
  • low porosity silica magnetic particles refers to silica magnetic particles with a median particle size of about lO ⁇ m to about 20 ⁇ m and a surface area, as measured by nitrogen BET method, of less than about 10 m 2 /g of particle mass.
  • DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage.
  • an end of an oligonucleotides or of a polynucleotide referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends.
  • the terms, "complementary” or “complementarity” are used herein in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. Specifically, adenosine (“A”) bases pair with thymidine (“T”) bases, and guanosine (“G”) bases pair with cytidine (“C”) bases on oppositely oriented polynucleotides. For example, the sequence “5'-AGT-3',” is complementary to the sequence "3'-TCA-5 ⁇ "
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the melting temperature of the formed hybrid, and the G:C ratio within the nucleic acids.
  • An oligonucleotide primer can be hybridized to a DNA template to initiate a DNA sequencing reaction or to amplify DNA, e.g., in a polymerase chain reaction.
  • oligonucleotide as used herein is a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than five, and usually ten or more. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotiede.
  • the oligonucleotide may be generated in any manner, including, but not limited to, chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligonucleotide, more preferably an oligo- deoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • purified or “to purify” means a process or the result of any process which removes some contaminants from the component of interest, such as a DNA extension product. The percent of a purified component is thereby increased in the sample.
  • a DNA extension product is purified from a dideoxy DNA sequencing reaction prior to analysis of the DNA extension product, using silica magnetic particles.
  • the sequencing DNA extension products are selectively adsorbed to the particles in an adsorption solution, thereby forming a complex, preferably leaving material in solution that might interfere with the analysis of the sequencing extension products (e.g. fluorescently-labeled dideoxy-terminators or primers).
  • the complex is subsequently separated from the adsorption solution.
  • the kit of the present invention provides silica magnetic particles and an adsorption solution designed for use in practicing the method of the present invention.
  • the method and kit of the present invention are suitable for use in purifying a DNA extension product from a dideoxy-terminated DNA sequencing reaction wherein either the primer or the dideoxy-nucleotide(s) has been labeled with either a radioactive label (e.g., P 32 or S 35 ) or with a fluorescent dye, or when a radiolabeled nucleotide is incorporated internally in the sequence.
  • the label is preferably a fluorescent dye, preferably a fluorescent dye with a fluoroscein donor dye linked to a rhodamine acceptor dye, such as FAM, JOE, TAMARA, or ROX.
  • the label is more preferably a fluorescent dye with a fluorescein donor dye linked to a dichlororhodamine acceptor dye, even more preferably, a fluorescent dye selected from the group consisting of: dichloro[R6G], dichloro[ROX], dichloro[R110], dichloro[TAMRA].
  • a fluorescent dye selected from the group consisting of: dichloro[R6G], dichloro[ROX], dichloro[R110], dichloro[TAMRA].
  • the fluorescein/dichlororhodamine dyes attached to deoxynucleotides or dideoxynucleotides available from ABI under the brand name Big Dye ® deoxynucleotides and BigDye® terminators respectively, are preferred over the fluorescent rhodamine dyes listed above because of their narrower and brighter emission spectra, giving less spectral overlap, and less noise.
  • the present method also separates the extension products from salts and other components of the extension reaction, which can have a deleterious effect on the analysis of the extension products.
  • the DNA extension products are preferably adsorbed to the silica magnetic particles, leaving the labeled primers in solution.
  • the silica magnetic particle used in the method and kit of the present invention can be macro-porous or of low porosity.
  • the silica magnetic particle is most preferably of low porosity. Macro-porous particles tend to be smaller than low porosity particles. However, due to the porosity of the macro-porous particles, the total surface area of a macro-porous particle is greater (on a per weight unit basis) than one would find on larger low porosity silica magnetic particles. Smaller particles are limited in the amount of magnetic material which can be incorporated into such particles compared to larger particles. Thus, even though the surface area of macro-porous particles is greater, low porosity particles can incorporate a greater amount of magnetic material, making them easier to separate from a solution in the presence of a magnetic field.
  • the median particle size of macro-porous silica magnetic particles used in the present invention is preferably about 1 to 10 ⁇ m, more preferably about 3 to 10 ⁇ m, and most preferably about 4 to 7 ⁇ m.
  • the median particle size of low porosity particles is preferably about 10 to 20 ⁇ m, more preferably about 12 to 18 ⁇ m, and most preferably about 13 to about 16 ⁇ m.
  • the particle size distribution may also be varied. However, a relatively narrow monodal particle size distribution is preferred.
  • the monodal particle size distribution is preferably such that about 80% by weight of the particles are within two standard deviations of the median particle size, more preferably within one standard deviation of the median particle size.
  • the siliceous oxide coating of each of the macro-porous silica magnetic particles used in the present invention has a total pore volume, as measured by nitrogen BET method, of at least about 0.2 ml/g, more preferably about 0.5 to 1.5 ml/g based on the total mass of the particles.
  • a total pore volume measured by nitrogen BET preferably at least about 50%o is contained in pores having a diameter of 600 A or greater, more preferably at least about 60%, most preferably about 70 to 85%.
  • the total surface area of a macro-porous silica magnetic particle is preferably at least about 10 m 2 /g, more preferably at least 20 m 2 /g, even more preferably at least 35 m 2 /g of particle mass, most preferably about 40 m 2 /g.
  • the total surface area of a macro-porous silica magnetic particle is preferably no more than about 500 m 2 /g, more preferably no more than about 100 m 2 /g, even more preferably no more than about 80 m 2 /g.
  • the total surface area of a low porosity silica magnetic particle is preferably less than about 10 m 2 /g, more preferably less than about 5 m /g, and more preferably less than about 2 m /g, and even more preferably less than about 0.05 m 2 /g.
  • Silica magnetic particles can contain substances, such as transition metals or volatile organics, which could adversely affect the utility of nucleic acids isolated using the particles, when the nucleic acids be become substantially contaminated with such substances. Specifically, such contaminants could adversely affect analysis of the sequencing extension products, after purification using the particles, according to the method of the present invention. Any such substances present in the silica magnetic particles used in the method and kit of the present invention are preferably present in a form that does not readily leach out of the particle and into the purified DNA sequencing reaction produced according to the method or using the kit of the present invention. Iron is one such potential contaminant.
  • Iron in the form of magnetite, is present at the core of particularly preferred forms of silica magnetic particles used in the method and kit of the present invention.
  • Iron has a broad absorption peak between 260 and 270 nanometers ("n ").
  • Nucleic acids, such as DNA have a peak absorption at about 260 nm, so iron contamination in a nucleic acid sample can adversely affect the accuracy of the results of quantitative spectrophotometric analysis of such samples.
  • Any iron containing silica magnetic particles used to isolate DNA sequencing extension products using the present invention preferably do not produce isolated extension products sufficiently contaminated with iron for the iron to interfere with spectrophotometric analysis of the material at or around 260 nm.
  • the most preferred macro-porous silica magnetic particles used as in the methods and kits of the present invention are siliceous oxide coated particles produced as described in international patent application publication number WO 98/31461.
  • the macro-porous silica magnetic particle production method disclosed therein can also be modified and adapted for use in producing the low porosity silica magnetic particles used in the present invention.
  • Low porosity silica magnetic particles suitable for use in the present invention are commercially available from WR Grace (Catalog No. MP-85). The description of the silica magnetic particles contained in WO 98/31461 is incorporated by reference herein.
  • the preferred silica magnetic particles leach no more than 50 ppm, more preferably no more than 10 ppm, more preferably no more than 7 ppm, and most preferably no more than 5 ppm of transition metals when assayed as described immediately below.
  • Transition metals from the particles Leaching of transition metals from the particles is assayed as follows: 0.33 g of the particles (oven dried @110°C) are combined with 20 ml. of IN HC1 aqueous solution (using deionized water). The resulting mixture is then agitated only to disperse the particles. After about 15 minutes total contact time, a portion of the liquid from the mixture is then analyzed for metals content. Any conventional elemental analysis technique may be employed to quantify the amount of transition metal in the resulting liquid, but inductively coupled plasma spectroscopy (ICP) is preferred. Macro-porous silica magnetic particles that meet the above-cited specification for particularly preferred particles are sold under the brand name MagneSilTM Paramagnetic Particles (Promega Corporation). Low porosity silica magnetic particles that meet the above-cited specification are available from WR Grace (Catalog No. MP-85).
  • ICP inductively coupled plasma spectroscopy
  • the adsorption solution used in the method and included in the kit of the present invention is configured to promote selective adsorption of DNA extension products to silica magnetic particles, when combined with the products of a dideoxy DNA sequencing reaction.
  • the adsorption solution preferably comprises a chaotropic agent and a buffer having a pH of less than about 7.0, preferably having a pH of less than about 6.0, more preferably having a pH of less than about 5.0.
  • the buffer is preferably an acetate buffer or a citrate buffer, more preferably a citrate buffer, most preferably sodium citrate.
  • the chaotropic agent is preferably, sodium iodide, potassium iodide, urea, sodium perchlorate, or a guanidine salt, more preferably guanidine hydrochloride or guanidine thiocyanate.
  • any one of a number of different means can be used to separate the complex from the adsorption solution.
  • Suitable separation means include, but are not limited to, vacuum or gravity filtration, decantation, centrifugation, or magnetic force.
  • the complex is most preferably separated from the adsorption solution in the presence of a magnetic force. Any source of magnetic force sufficiently strong to separate the silica magnetic particles from a solution would be suitable for use in separating the complex from the adsorption solution and from other solutions used in preferred additional steps of the present method.
  • the magnetic force is preferably provided in the form of a magnetic separation stand, such as one of the MagneSphere ® Technology Magnetic Separation Stands (Cat. Nos. Z5331 to 3, or Z5341 to 3) from Promega Corporation.
  • the method of the present invention preferably further comprises a step of washing the complex prior to elution of the DNA extension products therefrom.
  • the composition of any wash solution used to wash the complex is selected to ensure the DNA extension products remain part of the complex.
  • the wash solution preferably comprises at least 50% of a low molecular weight alcohol, such as ethanol or isopropanol, more preferably at least 60% ethanol, even more preferably at least 70% ethanol, even more preferably at least 90% ethanol.
  • the nucleic acid adsorption solution and any wash solution are preferably prepared from or consist of distilled or deionized water.
  • the distilled, deionized, or other type of water used in the nucleic acid adsorption solution is preferably filtered prior to use, using a filtration that achieves at least 18 mega Ohms-resistance of water.
  • the Nanopure ® Filtration System (Bamestead) can be used to produce such water.
  • the distilled, deionized, or filtered water can be autoclaved prior to use in the method or in the kit of the present invention.
  • Elution of a nucleic acid, such as a DNA extension product, from the complex is carried out in the presence of an elution solution selected for its capacity to ensure the release of the extension product from the complex.
  • the elution solution preferably comprises a component selected from the group consisting of water, formamide, and a tracking dye.
  • elution solution comprises a tracking dye it is preferably a dye suitable for use in tracking the DNA extension products as they are fractionated by gel or capillary electrophoresis.
  • the elution solution is most preferably a loading solution, containing all the components necessary for loading a sample of the DNA extension products onto either an electrophoresis gel or a capillary electrophoresis capillary.
  • the elution solution preferably has a pH of at least about 5.0 and up to about 8.0, more preferably at least about 6.0 and up to about 8.0.
  • the DNA extension products are preferably analyzed by gel or capillary electrophoresis.
  • the purified reaction produces results with low backgrounds and high accuracy of read, even close to the primer.
  • Read is considered, herein, to begin at the point closest to a primer in which four bases in a row are accurately read. Thereafter, read tends to be accurate.
  • Fluorescent dye labeled DNA extension products isolated according to the present invention produce at least 98% of readable sequence when a sequence is read out to 600 bases, with readable sequence beginning within 50 bases after the primer sequence, more preferably producing at least 99% of readable sequence out to 600 bases, with readable sequence beginning within 25 bases, even more preferably with readable sequence beginning within 10 bases after the primer sequence.
  • the MagneSilTM particles used in the Examples below were taken from either of two batches of particles having the following characteristics: (1) a BET surface area of 55 m 2 /g, pore volume of 0.181 ml/g for particles of ⁇ 600 A diameter, pore volume of 0.163 ml g for particles of >600 A diameter, median particle size of 5.3 ⁇ m, and iron leach of 2.8 ppm when assayed as described herein above using ICP; or (2) a BET surface area of 49 m 2 /g, pore volume of 0.160 ml g ( ⁇ 600 A diameter), pore volume of 0.163 ml g (>600 A diameter), median particle size of 5.5 ⁇ m, and iron leach of 2.0 ppm.
  • EXAMPLE 1 Purification of a Dye Primer Sequencing Reaction ⁇ GEM ® -3Zf+ DNA template (Promega Cat. #P227) was sequenced, using a Dye Primer DNA sequencing kit from ABI (Cat. # 402112) and the cycling conditions listed in Table 2 below the Wizard® DNA Purification products (i.e., Promega, Cat. #A7100).
  • thermocycler was heated prior to use. The samples were not placed into the thermocycler until the block temperature had reached 85° C. The entire thermocycling program took about 1 hour and 15 minutes.
  • the sequencing reaction was divided into twelve tubes (30 ⁇ l each) and treated in 12 different ways to remove the dye primers prior to running the sequence on an ABI PRISM ® 377 DNA Sequencer.
  • the reactions in tubes one and two were ethanol ("EtOH") precipitated by combining with 80 ⁇ l chilled 95% ethanol and incubating on ice for 15 minutes.
  • the tubes were then centrifuged at 14,000 rpm in a microcentrifuge for 30 minutes at 4°C.
  • the supernatant was removed and the DNA pellet washed with 100 ⁇ l chilled 70% ethanol.
  • the tubes were again centrifuged at 14,000 rpm in a microcentrifuge for 5 min at 4°C.
  • the supematant was removed, the pellet dried and resuspended in 6 ⁇ l loading dye (5 parts deionized formamide: 1 part EDTA/Blue Dextran).
  • the reactions in tubes 3-12 were treated with MagneSilTM particles in various binding buffers to remove the dye primers.
  • MagneSilTM particles 45 ⁇ l were equilibrated in 600 ⁇ l of an adsorption solution consisting of the concentration of guanidine thiocyanate ("GTC") given in Table 4, below, and 0.08 M potassium acetate (“KOAc”), pH 4.8, and washed three times in 600 ⁇ l of the adsorption solution.
  • GTC guanidine thiocyanate
  • KOAc potassium acetate
  • Samples 3-12 were processed as follows, prior to analysis: 200 ⁇ l of each of the MagneSilTM particles in binding buffer was added to an aliquot of the sequencing reaction, allowed to bind for one minute, magnetized, and the supernatant removed.
  • the pellets of MagneSilTM particles for samples 3-7 were washed twice in 70% ethanol, while the pellets for samples 8-12 were washed twice in 80% ethanol. After the final wash was removed the pellets were allowed to air dry for 5 minutes and each resuspended in 50 ⁇ l water. Then 200 ⁇ l of the appropriate binding buffer, as used initially with the particles, was added to each pellet, the pellet magnetized, and the supernatant removed. The pellets were washed with ethanol as described above.
  • Table 5 below, provides a summary of the results of analyzing the samples purified, as described above, on an ABI 377 DNA Sequencer.
  • the percent accuracy results in Table 5 are percent accuracy in 600 bases read.
  • the final column of data in Table 5 shows the number of bases from the primer at which readable sequence began.
  • Example 2 Comparison of Ethanol Precipitation to Use of MagneSilTM Particles in Purifying a Dye Terminator Labeled Sequencing Reaction.
  • This example reflects the sequencing reaction using BigDyeTM Terminator Sequencing conditions with wash conditions using either ethanol precipitation or MagneSilTM particles (Promega, part #) in 0.6 M GTC/ 0.08 M KOAc adsorption solution as described in Example 1.
  • pGEM ® -3Zf DNA template (Promega, P227) was sequenced using a primer having the sequence identified by SEQ ID NO: 2 (5' GTTTTCCCAGTCACGAC 3') and the ABI PRISM® BigDyeTM Terminator Sequencing Chemistry (Perkin Elmer Catalog No.
  • a 7X master mix of BigDyeTM Te ⁇ ninator sequencing reaction was assembled by combining 56 ⁇ l Terminator Ready Reaction Mix (dideoxy-A labeled with dichloro [R6G], dideoxy-C labeled with dichloro [R0X], dideoxy-G labeled with dichloro [R110], dideoxy- T labeled with dichloro[TAMRA], a mixture of dATP, dCTP, dITP, and dUTP, AmpliTaq DNA Polymerase FS, thermally stable pyrophosphatase, MgCl 2 , and Tris buffer, pH 9.0) 17.5 ⁇ l of template DNA (200 ng/ ⁇ l), 12.6 ⁇ l Primer 1 (10 ⁇ g/ ⁇ l), and 53.9 ⁇ l water.
  • the resulting sequencing reactions were then either ethanol precipitated or treated with MagneSilTM particles to remove the BigDye Terminators.
  • the ethanol precipitation was performed on samples 1 and 2 by adding 16 ⁇ l water and 64 ⁇ l room temperature 95% ethanol to the sequencing reaction.
  • the tubes were incubated at room temperature for 15 minutes and then centrifuged at 14,000 rpm in a microcentrifuge for 20 min at 4°C. The supernatant was removed and the DNA pellet washed with 250 ⁇ l room temperature 70% ethanol.
  • the tubes were then centrifuged at 14,000 rpm in a microcentrifuge for 10 min at 4°C. The supernatant was removed and the pellet was vacuum dried for 15 minutes. Each dried pellet was resuspended in 6 ⁇ l loading dye.
  • Samples 3-6 were purified using MagneSilTM particles, as follows. First, 37.5 ⁇ l MagneSilTM particles were mixed with 1 ml binding buffer (0.6 M GTC / 0.08 M KOAc) as described in example 1. Then 200 ⁇ l of the MagneSilTM particles in the binding buffer were added to each sample and allowed to bind for 5 minutes at room temperature. The tubes were gently mixed to keep the particles in solution. The particles were magnetized and the supernatant removed. The particle pellets were washed once with 200 ⁇ l 80% ethanol and remagnetized. The supernatants were removed and the pellets air-dried for 10 minutes.
  • 1 ml binding buffer 0.6 M GTC / 0.08 M KOAc
  • Example 3 MagneSilTM Particles in Modified BigDyeTM Terminator Purification
  • BigDyeTM Terminators were removed from a sequencing reaction with conditions much like those described in Example 2 for MagneSilTM Particles.
  • the difference illustrated in this example is that the adsorption solution was 1 M GTC, 80 inM KOAc at pH 4.8 and the wash solution was 90% ethanol.
  • the BigDyeTM Terminator chemistry was performed as recommended by the manufacturer with the exception that a 1:4 dilution of the terminator ready reaction mix was used. See above example for the formulation of this mix
  • This example compared three methods for removal of BigDyeTM Terminators from sequencing reactions.
  • the methods used were 1) ethanol precipitation, 2) MagneSilTM particles, and 3) Low-porosity magnetic particles.
  • the sequencing reactions were performed with the ABI PRISM ® BigDyeTM Terminator Sequencing System according to manufacturer's instruction with the template and primer described above in Example 2.
  • the termination ready mix of the sequencing system was either not diluted, 1:4 diluted, or 1:8 diluted to represent conditions often utilized by end-users of this system.
  • the amplification conditions were previously described in Example 2.
  • the sequencing reactions (20 ⁇ l) purified by ethanol precipitation were each combined with 16 ⁇ l nanopure water and 64 ⁇ l room temperature 95% ethanol.
  • the reactions were incubated at room temperature for 15 minutes and then centrifuged at 14,000 rpm in a microcentrifuge for 20 minutes at 4°C.
  • the supematants were removed and the pellets washed with 250 ⁇ l room temperature 70% ethanol.
  • the tubes were centrifuged at 14,000 rpm in a microcentrifuge for 10 minutes at 4°C.
  • the supematants were removed and the pellets vacuum-dried for 15 minutes.
  • the pellets were resuspended in 10 ⁇ l loading dye and 1.5 ⁇ l run on an ABI 377 Sequencer as described in Example 2.
  • Magnetic particles were prepared by mixing 45 ⁇ l MagneSilTM particles (porous) in
  • 1080 ⁇ l binding buffer and by mixing 45 ⁇ l Low-porosity magnetic particles in 1080 ⁇ l binding buffer.
  • the binding buffer was 0.6 M GTC / 0.08 M KOAc for both types of particles.
  • the porous particles were 100 mg/ml, the low-porosity particles were 133 mg/ml.
  • 180 ⁇ l of the particles inding buffer mixture was added to the appropriate reactions as listed below. They were allowed to bind for 5 minutes with frequent mixing of the tubes.
  • the tubes were then placed on a magnet, the particles allowed to collect against the tube and the supematants removed.
  • the pellets were washed once with 200 ⁇ l 80% ethanol and the supematants removed.
  • the pellets were allowed to air dry and the nucleic acid eluted in 10 ⁇ l loading dye for 1 minute, magnetized, and 1.5 ⁇ l of the supernatant run on an ABI 377 Sequencer as described in Example 2.
  • the samples were loaded and run on the sequencer as described in Table 7, below, producing the results shown in the final columns of the table:
  • EXAMPLE 5 Use of Low-Porosity Silica Magnetic Particles for BigDyeTM Terminator Purification
  • the five batches of low porosity particles described in Table 1, above, were combined and divided into four lots, A, B, C, and D, that were tested in triplicate to remove the terminators. Twenty microliters of sequencing reaction was added to the following mixture:
  • the tubes were incubated at room temperature for 5 minutes with periodic mixing. The particles were magnetized and the supernatant was removed. The particles were then washed twice, 5 minutes each wash, with 100 ⁇ l 90% ethanol. The particles were then magnetized and the supernatant removed. The particles were allowed to air dry for 15 minutes and then resuspended in loading dye and run on and ABI 377 sequencer.
  • the sequencing reaction was purified by ethanol precipitation prior to running on the sequencer.
  • a 20 ⁇ l sample was added 16 ⁇ l water and 64 ⁇ l 95% ethanol.
  • the tube was incubated at room temperature for 15 minutes and then centrifuged at 11,000 rpm in a microcentrifuge for 20 minutes at 4°C. The supernatant was removed and the pellet washed in 70% ethanol. The pellet was dried in a vacuum and resuspended in loading dye.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un kit permettant d'utiliser des particules magnétiques de silice, de préférence des particules magnétiques de silice à faible porosité, afin de purifier des produits d'extension d'ADN à partir de réactions de séquençage d'ADN contenant dans la solution des didéoxynucléotides non incorporés et d'autres matières qui sont susceptibles d'interférer avec l'analyse des séquences, avant le début de celle-ci. Dans un mode de réalisation préféré de cette invention, on a conçu le procédé et le kit pour les utiliser dans la purification des produits d'extension d'ADN à partir de réactions de séquençage d'ADN contenant dans la solution des didéoxynucléotides non incorporés étiquetés avec des colorants fluorescents et d'autres matières. Selon la présente méthode, les produits d'extension d'ADN purifiés à partir d'une réaction de séquençage ou utilisant le kit susmentionné produisent des lectures extrêmement précises et longues comparées aux réactions purifiées utilisant d'autres méthodes.
EP01998656A 2000-11-28 2001-11-21 Purification de reactions de sequen age d'adn au moyen de particules magnetiques de silice Withdrawn EP1339876A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US72416900A 2000-11-28 2000-11-28
US724169 2000-11-28
PCT/US2001/043364 WO2002044414A2 (fr) 2000-11-28 2001-11-21 Purification de reactions de sequençage d'adn au moyen de particules magnetiques de silice

Publications (1)

Publication Number Publication Date
EP1339876A2 true EP1339876A2 (fr) 2003-09-03

Family

ID=24909311

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01998656A Withdrawn EP1339876A2 (fr) 2000-11-28 2001-11-21 Purification de reactions de sequen age d'adn au moyen de particules magnetiques de silice

Country Status (3)

Country Link
EP (1) EP1339876A2 (fr)
AU (1) AU2002217784A1 (fr)
WO (1) WO2002044414A2 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7875440B2 (en) 1998-05-01 2011-01-25 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US6780591B2 (en) 1998-05-01 2004-08-24 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
GB0207495D0 (en) 2002-03-28 2002-05-08 Biochemie Gmbh Organic compounds
GB0215185D0 (en) 2002-07-01 2002-08-07 Genovision As Binding a target substance
US7759055B2 (en) 2002-08-28 2010-07-20 Millipore Corporation Compositions of solution for sequencing reaction clean-up
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
DE602005020421D1 (de) 2004-02-19 2010-05-20 Helicos Biosciences Corp Verfahren zur analyse von polynukleotidsequenzen
US7378260B2 (en) * 2005-04-01 2008-05-27 Applera Corporation Products and methods for reducing dye artifacts
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US20170051344A1 (en) 2014-01-24 2017-02-23 Life Technologies Corporation Purification Chemistries and Formats for Sanger DNA Sequencing Reactions on a Micro-Fluidics Device
CN107236726A (zh) * 2017-05-23 2017-10-10 北京创新乐土基因科技有限公司 Clean‑CL纯化试剂盒及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4233169A (en) * 1979-04-13 1980-11-11 Corning Glass Works Porous magnetic glass structure
US5683875A (en) * 1995-05-04 1997-11-04 Hewlett-Packard Company Method for detecting a target nucleic acid analyte in a sample
US6027945A (en) * 1997-01-21 2000-02-22 Promega Corporation Methods of isolating biological target materials using silica magnetic particles
US6194562B1 (en) * 1998-04-22 2001-02-27 Promega Corporation Endotoxin reduction in nucleic acid purification

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0244414A3 *

Also Published As

Publication number Publication date
WO2002044414A2 (fr) 2002-06-06
WO2002044414A3 (fr) 2003-05-22
AU2002217784A1 (en) 2002-06-11

Similar Documents

Publication Publication Date Title
AU774810B2 (en) Mixed-bed solid phase and its use in the isolation of nucleic acids
US6787307B1 (en) Lysate clearance and nucleic acid isolation using silanized silica matrices
US8569477B2 (en) Method for isolating nucleic acids comprising the use of ethylene glycol multimers
EP1448799B2 (fr) Procédés d'isolation selective d'acides nucleiques
US7022835B1 (en) Method for binding nucleic acids to a solid phase
AU771249B2 (en) Method for purification and manipulation of nucleic acids using paramagnetic particles
JP2703636B2 (ja) 核酸を精製する方法
EP1173559A1 (fr) Procede de purification d'acides nucleiques a l'aide de carbure de silicium
US20110172409A1 (en) Method for nucleic acid isolation by solid phase reversible binding of nucleic acids
WO2001025490A1 (fr) Retrait de codons d'arret didesoxy marques par colorant provenant de reactions de sequençage d'adn
WO2002044414A2 (fr) Purification de reactions de sequençage d'adn au moyen de particules magnetiques de silice
AU2002225942B2 (en) Lysate clearance and nucleic acid isolation using silanized silica matrices
JPH05125088A (ja) Ｄｎａの固相抽出精製
AU2002225942A1 (en) Lysate clearance and nucleic acid isolation using silanized silica matrices
US20040121336A1 (en) Method for generating multiple samples containing a predetermined amount of nucleic acid
JP4764966B2 (ja) メッセンジャーｒｎａの分離方法
JPH11196869A (ja) リボ核酸の単離方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030627

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20041014