Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-03-20
2004-10-19
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025410, C536S018500, C435S006120
Reexamination Certificate
active
06806362
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
TECHNICAL FIELD
This invention relates generally to materials and methods for isolating a target nucleic acid, such as plasmid DNA, chromosomal DNA, total RNA, mRNA, or RNA/DNA hybrids from contaminants, such as proteins, lipids, cellular debris, and non-target nucleic acids. This invention relates, particularly, to pH dependent ion exchange matrices with the capacity to adsorb a target nucleic acid in the presence of a solution at a first pH and to desorb the target nucleic acid in the presence of a second solution at a second pH which is different from the first pH. This invention also relates to methods of making and using such pH dependent ion exchange matrices in isolating target nucleic acids.
BACKGROUND OF THE INVENTION
Many molecular biological techniques such as reverse transcription, cloning, restriction analysis, amplification and sequencing require that nucleic acids used in the techniques be substantially free of contaminants capable of interfering with such processing or analysis procedures. Such contaminants generally include substances that block or inhibit chemical reactions, (e.g. substances that block or inhibit nucleic acid hybridizations, enzymatically catalyzed reactions and other types of reactions used in molecular biological techniques), substances that catalyze the degradation or depolymerization of a nucleic acid or other biological material of interest, or substances which block or mask detection of the nucleic acid of interest. Substances of this last type can block or mask by providing a “background” indicative of the presence in a sample of a quantity of a nucleic acid of interest, (also referred to herein as a “target nucleic acid”) when the nucleic acid of interest is not, in fact, present in the sample. Contaminants also include macromolecular substances from the in vivo or in vitro medium from which a target nucleic acid is isolated, macromolecular substances such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors, oligonucleotides, or non-target nucleic acids. Contaminants can also be introduced into a target biological material from chemicals or other materials used to isolate the material from other substances. Common contaminants of this last type include trace metals, dyes, and organic solvents.
Obtaining target nucleic acid sufficiently free of contaminants for molecular biological applications is complicated by the complex systems in which the target nucleic acid is typically found. These systems, e.g., cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the target nucleic acid of interest must be isolated before being used in a molecular biological procedure.
The earliest techniques developed for use in isolating target nucleic acids from such complex systems typically involve multiple organic extraction and precipitation steps. Hazardous chemicals, such as chloroform and phenol or mixtures thereof, were used in most such procedures. Closed circular nucleic acid molecules, such as plasmid DNA, was typically isolated further by ultra-centrifugation of plasmid DNA in the presence of cesium chloride and ethidium bromide. See, e.g.,
Molecular Cloning
, ed. by Sambrook et al. (1989), pp. 1.42-1.50. Ethidium bromide is a neurotoxin. Removal of both ethidium bromide and cesium chloride from the resulting band of plasmid DNA obtained by ultracentrifugation was required before the DNA could be used in downstream processing techniques, such as sequencing, transfection, restriction analysis, or the polymerase chain reaction.
In recent years, many different matrices have been developed for use in the isolation of nucleic acids from complex biological materials. For example, matrices have been developed for the isolation of nucleic acids by ion-exchange chromatography (e.g.,
J. of Chromatog.
508:61-73 (1990);
Nucl. Acids Research
21(12):2913-2915 (1993); U.S. Pat. Nos. 5,856,192; 5,82,988; 5,660,984; and 4,699,717), by reversed phase (e.g. Hirbayashi et al.,
J. of Chromatog.
722:135-142 (1996); U.S. Pat. No. 5,057,426, by affinity chromatography (e.g., U.S. Pat. No. 5,712,383; and PolyATract® mRNA Purification System (Promega Corp., Madison, Wis.; see Promega's Technical Manual No. TM031), and by matricies which employ a combination of the above isolation modes (see, e.g. U.S. Pat. No. 5,652,348;
J. Chromatography
270:117-126(1983))
One of the first solid phases developed for use in isolating nucleic acids was a specialized resin of porous silica gel particles designed for use in high performance liquid chromatography (HPLC). The surface of porous silica gel particles was functionalized with anion-exchangers which could exchange with plasmid DNA under certain salt and pH conditions. See, e.g. U.S. Pat. Nos. 4,699,717, and 5,057,426. Machrey-Nagel Co. (Düren, Germany) was one of the first companies to provide HPLC columns packed with such anion-exchange silica gel particles, and it continues to sell such columns today. See, e.g. Information about NUCLEOGEN® 4000-7DEAE in product information downloaded from the Machrey-Nagel homepage on the Internet on Jun. 12, 1998, at http://www.machrey-nagel.com. Each such column was designed so that plasmid DNA bound thereto is eluted in an aqueous solution containing a high concentration of a highly corrosive salt (e.g. plasmid DNA is eluted from the NUCLEOGEN® 4000-7DEAE column in 6 M urea). Each such column had to be washed thoroughly between each isolation procedure to remove the corrosive salt and contaminants bound to the column with the DNA from the system. The nucleic acid solution eluted therefrom also had to be processed further to remove the corrosive salt therefrom before it could be used in standard molecular biology techniques, such as cloning, transformation, digestion with restrictive enzymes, or amplification.
Various silica-based solid phase separation systems have been developed since the early HPLC systems described above. (See, e.g. the silica gel and glass mixture for isolating nucleic acids according to U.S. Pat. No. 5,658,548, and the porous support with silane bonded phase used to isolate oligonucleotides according to U.S. Pat. No. 4,767,670.) Modem silica-based systems utilize 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 modem 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. Such 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. Numerous commercial sources offer silica-based resins designed for use in centrifugation and/or filtration isolation systems. See, e.g. Wizard® DNA purification systems products from Promega Corporation (Madison, Wis., U.S.A.); or the QiaPrep® DNA isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.)
Magnetically responsive particles, formerly used to isolate and purify polypeptide molecules such as proteins or antibodies, have also been developed for use as solid phases in isolating nucleic acids. Several different types of magnetically responsive particles designed for isolation of such materials are described in the literature, and many o
Bitner Rex M.
Grosch Josephine C.
Holmes Diana L.
Katzenhendler Jehoshua
Simpson Daniel J.
Frenchick Grady J.
Marschel Ardin H.
Michael & Best & Friedrich LLP
Promega Corporation
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