Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-13
2004-09-07
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007100, C435S007200
Reexamination Certificate
active
06787307
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
TECHNICAL FIELD
This invention relates generally to methods of using modified silica matrices to clear solutions of disrupted biological material, such as cell lysates or homogenates of plant or animal tissue. This invention also relates to the use of such matrices to isolate target nucleic acids, such as plasmid DNA, chromosomal DNA, DNA fragments, total RNA, mRNA, or RNA/DNA hybrids from non-target material, such as proteins, lipids, cellular debris, and non-target nucleic acids. This invention relates, particularly, to the use of silanized silica matrices in lysate clearance and in target nucleic acid isolation.
BACKGROUND OF THE INVENTION
Various methods have been developed for isolating target nucleic acids from biological material or from other types of material containing the nucleic acids. When the target nucleic acid is contained in the interior of a cell, the cell membrane must be disrupted and the contents of the cell released into the solution surrounding the cell before the target nucleic acid can be isolated from other cellular material. Such disruption can be accomplished by mechanical means (e.g., by sonication or by blending in a mixer), by enzymatic digestion (e.g., by digestion with proteases), or by chemical means (e.g., by alkaline lysis followed by addition of a neutralization solution). Whatever means is used to disrupt a cell, the end product, referred to herein as a lysate solution, consists or the target material and many contaminants, including cell debris.
Centrifugation or filtration are commonly used to clear a lysate solution of as many of the large contaminants as possible before the target nucleic acid material is isolated therefrom. Unfortunately, neither filtration nor centrifugation is readily amenable to automation. Specifically, neither are typically performed at basic pipettor-diluter robotics stations, such as the Biomek®-2000 (Beckman Coulter, Inc.; Fullerton, Calif.).
Many materials and methods have been developed for use in the isolation of nucleic acids from cleared lysate solutions. One such method is extraction of a nucleic acid from an agarose gel after fractionation of the nucleic acid by gel electrophoresis. Known means of extraction of nucleic acids from gel slices include dialysis, solvent extraction, and enzymatic digestion of the agarose. Such systems of nucleic acid extraction from an agarose gel slice tend to be very labor-intensive, and not amenable to automation. Furthermore, smaller sized fragments of DNA or RNA (i.e., below about 100 base pairs) tend to be lost in the extraction process.
Other systems of nucleic acid extraction are silica based, such as 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. Numerous commercial sources offer silica-based resins designed for use in centrifugation and/or filtration isolation systems, 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.). Unfortunately, the type of silica-based solid phases described above all require one to use centrifugation or filtration to perform the various isolation steps in each method, limiting the utility of such solid phases in automated systems.
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 he 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 MagneSil™ Paramagnetic Particles (Promega), or BioMag® Paramagnetic Beads (Polysciences, Warrington, Pa., U.S.A.). See also U.S. Pat. No. 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. (Lincoln Park, N.J. U.S.A.), or porous magnetic glass particles described in U.S. Pat. Nos. 4,395,271; 4,233,169, or 4,297,337. Nucleic acid material tends to bind very tightly to glass, however, so that it can be difficult to remove nucleic acids from such magnetic glass particles, once bound thereto. As a result, elution efficiencies from magnetic glass particles tend to be low compared to elution efficiencies from particles containing lower amounts of a nucleic acid binding material such as silica.
A variety of silica matrices have also been developed which consist of a silica solid phase with ligands covalently attached thereto designed to participate in ion exchange or in reversed-phase interaction with nucleic acids. However, such systems are generally designed for use as a solid phase of a liquid chromatography system, for use in a filtration system, or for use with centrifugation to separate the solid phase from various solutions. Such systems range in complexity from a single species of ligand covalently attached to the surface of a filter, as in DEAE modified filters (e.g., CONCERT® isolation system. Life Technology Inc., Gaithersburg, Md., U.S.A.), to a column containing two different solid phases separated by a porous divider (e.g., U.S. Pat. No. 5,660,984), to a chromatography resin with pH dependent ionizable ligands covalently attached thereto (e.g., U.S. Pat. No. 5,652,348), to mixed-mode or mixed-bed resins with ion exchange ligands and reversed-phase ligands on the same or on different solid phase components of the resins, respectively (e.g., McLaughlin, L. M.,
Chem Rev
(1989) 89:309-319).
Matrices have also been developed which are designed to reversibly bind to specific target materials through affinity interaction. Some such matrices use affinity of the poly (A) tail of mRNA for oligo (dT) to isolate mRNA, either by attaching oligo (dT) directly to the surface of a solid phase (e.g., U.S. Pat. No. 5,610,274), or by providing a solid phase coated with streptavidin and biotinylated oligo (dT) which naturally binds to both the streptavidin and to mRNA in a solution (e.g., PolyATract® Series 9600™ mRNA Isolation System from Promega Corporation (Madison, Wis., U.S.A.); and ProActive® streptavidin coated microsphere particles from Bangs Laboratories (Carmel, Ind., U.S.A.)).
Silanization has been used as a coupling agent to facilitate the covalent attachment of various ligands to the silica solid phases to produce chromatographic matrices for the isolation of solutes, such as nucleic acids. See, e.g., U.S. Pat. No. 4,672,040 (col. 13, lines 3-22); U.S. Pat. Nos. 5,734,020; 4,695,392; and 5,610,274). In such reactions a silane compound, such as 3-glycidoxypropyltrimethoxysilane, is reacted with the surface of a solid phase, such as a silica based material or with an iron oxide, such that the silane becomes attached thereto. The resulting matrix includes highly reacti
Bitner Rex M.
Flemming Roderick G.
Koller Susan C.
Simpson Daniel J.
Fahrlander Jill A.
Frenchick Grady J.
Michael & Best & Friedrich LLP
Promega Corporation
Riley Jezia
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