Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-02-22
2003-09-02
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S489000, C436S523000, C436S524000, C436S527000, C210S633000
Reexamination Certificate
active
06613895
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods and systems for rapidly isolating nucleic acids. More particularly, this invention contemplates systems and methods for isolating plasmid DNA from cell lysates and DNA from agarose gels.
BACKGROUND
The isolation of preparative amounts of biologically active nucleic acid molecules has been a vexing problem in molecular biology. This is especially the case with regard to isolation of DNA for use in recombinant methodologies where it is required to be in sufficiently pure form to be digestible by restriction endonucleases, to be a good substrate for polymerases and topoisomerases, and to be suitable for use as a transfection or transformation agent.
Over the years, many methods have been developed to isolate nucleic acid molecules. However, those methods are typically tedious, require a high level of skill to perform, take extended periods of time to accomplish, require the processing of relatively large volumes of materials and often give variable results. In addition, most of the isolation techniques reported are costly in terms of equipment and materials. See, Gamper et al.,
DNA
, 4:157-164 (1985); Yang et al.,
Meth. Enzol
., 68:176-182 (1979); and Vogelstein et al.,
Proc. Natl. Acad. Sci. USA
, 7:615-619 (1979).
To date, the art has applied several different technologies to the problem of preparing nucleic acids in quantities and purities sufficient for use in recombinant methodologies. Classically, the final step in the isolation of plasmid DNA is a cesium chloride-ethidium dye isopycnic gradient ultracentrifugation through a neutral or alkaline density gradient, gel electrophoresis, high-pressure liquid chromatography through RPC-5, alkaline extraction and column chromatography on methylated albumin kieselguhr, hydroxyapatite, or benzoylated naphthoylated DEAE-cellulose. See Gamper et al.,
DNA
, 4:157-164 (1985); Yang et al.,
Meth. Enzol
. 68:176-182 1979); Birnboim.
Meth. Enzol
., 100: 243-255 (1983); Mizutani. J.
Chrom
., 262:441-445 (1983); and the references cited therein.
Of particular interest to the present invention are methods wherein the nucleic acid to be isolated is adsorbed onto an insoluble silica matrix, e.g., particulate glass. While there are several reports of using the binding of nucleic acids to particulate glass as an isolation means, the physio-chemical mechanism(s) responsible for the binding phenomenon and the conditions under which it occurs are poorly characterized. Advances in the art have therefore proceeded on an empirical basis.
The use of adsorption onto glass as a means for isolating nucleic acids is based on the observation that both DNA and RNA bind to glass in highly concentrated aqueous salt solutions, i.e., salt concentrations of at least about 3 molar, and can be eluted therefrom by lowering the salt (ionic) concentration. While the pH value of the salt solution appears to have some effect on the adsorption process, that effect has not been characterized.
There have been several reports on the use of the glass-adsorption technique to isolate DNA from agarose gels. In each case, the salt solutions used to mediate the binding of the nucleic acids to the glass contained the buffering agent tris (hydroxy-methyl) aminomethane at a concentration of less than 50 millimolar. Those solutions therefore had a low buffering capacity. See, Mizutani,
J. Col. Inter. Sci
., 93:270-273 (1983); Mizutani,
J. Chrom
., 262:441-445 (1983); Marko et al.,
Anal. Biochem
., 121:382-387 (1982); Chen et al.,
Anal. Biochem
., 101:339-341 (1980); Vogelstein et al.,
Proc. Natl. Acad. Sci. USA
, 76:615-619 (1979); and Yang et al.,
Meth. Enzol
., 68:176-182 (1979).
None of the previously reported methods of isolating nucleic acids by glass-adsorption has gained widespread acceptance by those skilled in the art of recombinant DNA technology. This is probably due to the inability of those methods to consistently separate DNA from sample contaminants such as RNA, protein and agarose. For instance, Marko et al., supra, reported that the buffered salt solution used to mediate DNA binding to glass was required to contain the chelating agent cyclohexanediamine tetraacetate (CDTA) in order to prevent binding of tRNA to glass and co-purification of the tRNA with the plasmid DNA.
From the foregoing it can be seen that there has been a long felt need by those practicing recombinant DNA technology for a reliable, rapid method for isolating nucleic acid molecules.
BRIEF SUMMARY OF THE INVENTION
he present invention contemplates systems and methods for isolating nucleic acids. The systems and methods take advantage of solutions to the problems, discovered by the inventor, of insufficient buffering capacity and excessive glass particle heterogeneity.
In addition, the novel systems approach described herein permits a significant reduction in the level of skill and time required to produce isolated DNA.
In one embodiment, the present invention contemplates a system, in kit form, for isolating plasmid DNA from an aqueous sample. The system comprises, in separate containers, particulate glass and a buffered aqueous salt solution having a pH value in the range of 7 to 8. The solution contains:
a) a salt at a concentration of at least 3 molar, and
b) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that 0.1 to 1 molar tris(hydroxy-methyl)aminomethane or 0.1 to 1 molar phosphate ion would provide in solution.
Another aspect of the present invention is a system, in kit form, for isolating plasmid DNA from a sample. The system comprises, in separate containers:
a) particulate glass; and
b) a buffered aqueous salt solution having a pH value in the range of 7.2-7.8. The solution consists essentially of:
i) 2 M NaI,
ii) 2.6 M KBr, and
iii) 0.66 M tris(hydroxymethyl)aminomethane.
Also contemplated is a system, in kit form, for isolating nucleic acid molecules. The system comprises a composition comprising particulate glass having a sedimentation rate through still water at unit gravity in the range of about 0.001 to about 1.0 cm/min.
A further aspect of this invention is a system, in kit form, for isolating DNA from an aqueous sample. The system comprises, in separate containers, particulate glass and a buffered salt admixture which upon dissolution in a predetermined amount of distilled water provides a solution having a pH value in the range of 7 to 8. The buffered salt admixture contains:
a) a salt in an amount sufficient to provide a concentration of at least 3 molar upon said dissolution, and
b) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that provided by 0.1 to 1 molar aqueous tris(hydroxymethyl)aminomethane or 0.1 to 1 molar aqueous phosphate ion.
A further embodiment of this invention is a method for isolating plasmid DNA from an aqueous sample. The method comprises the steps of:
a) forming a binding reaction admixture by admixing said sample with an insoluble silica matrix and a buffered aqueous salt solution having a pH value in the range of 7 to 8, said solution containing i) a salt at a concentration of at least 3 molar, and ii) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that which 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar sodium phosphate would provide in said solution;
b) maintaining said binding reaction admixture for a time period sufficient for said DNA to bind to said matrix to form an insoluble DNA-matrix complex and a remaining admixture;
c) separating said remaining admixture and said complex to form an isolated complex; and
d) recovering said DNA from said isolated complex to form isolated plasmid DNA.
The present invention also contemplates a method for isolating DNA from an agarose gel sample. The method comprises the steps of:
a) forming a gel-dissolving reaction admixture by admixing said sample with a buffered aqueous chaotropic salt solution having a pH value in the range of 7 to 8, said solution containing i) a chaotropic s
Brolaski Mark
Gautsch James
BIO 101
Gray Cary Ware & Freidenrich LLP
Owens Howard V
Wilson James O.
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