Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-07-05
2003-01-07
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S127000, C521S030000
Reexamination Certificate
active
06504021
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is methods for purification of DNA.
BACKGROUND TO THE INVENTION
DNA purification technologies fall into two general categories, methods that involve selective adsorption of DNA to a substrate, and methods that involve removal of contaminants from soluble DNA. The latter was one of the first technologies used for the purification of DNA and involved primarily the extraction of DNA with liquid phenol, followed by precipitation with salt and ethanol (Maniatis, T., et al., Eds. Molecular Cloning, Cold Spring Harbor Lab., 90-91, 1982.). Additional variations on this approach have been developed including salting-out techniques that use protein precipitants as well as solid phase technologies that adsorb biological contaminants. These are disclosed in U.S. Pat. No. 5,561,064 and U.S. Pat. No. 4,923,978. The phenolic technology, while still in use, is time-consuming, the reagent is toxic and environmentally damaging and the technology is difficult to automate. The aforementioned variations on this technique tend to be expensive and time-consuming. For these and other reasons, selective adsorption of DNA to a substrate has become the most commonly used technology in the last decade. In the presence of chaotropic agents, DNA can be adsorbed to a variety of silicates as disclosed by Vogelstein, B., et al., (Proc. Natl. Acad. Sci. USA 76:615-619, Febuary 1979) and in U.S. Pat. Nos. 5,075,430 and 5,234,809 as well as in some non-silicate materials (U.S. Pat. No. 5,705,628). More recently, silicates and other silicon-containing substances have been developed that have reduced dependency on salt, as disclosed in U.S. Pat. Nos. 5,523,392 and 5,525,319). Once adsorbed, soluble contaminants are physically removed and co-adsorbed contaminants are removed by selective washing. The purified DNA is then eluted from the substrate with water or a weak buffer. DNA may also be purified by adsorption to ion exchange resins. DNA is a negatively charged molecule that binds when contacted with cationic resins. U.S. Pat. No. 5,057,426 discloses a method for purifying DNA with a porous cationic resin matrix. In this method, the DNA binds to the matrix and soluble contaminants are physically removed. Co-adsorbed contaminants are removed by selective washing with salt. Purified DNA is subsequently desorbed from the resin by high concentrations of salt. Due to the high density of negative charges on DNA, it is relatively easy to separate DNA from a variety of closely related contaminants and it is even possible to separate different types of DNA from each other. These selective adsorption methods have the advantage of providing high purity DNA in a relatively short period of time. However, as the number of applications increase and the demand for faster, cheaper, simpler and more automatable technology increases, the need for better technologies or improvements to existing technologies becomes apparent. In this application, we describe a novel method for purifying DNA that is rapid, simple and highly automatable. This method is based on the observation that DNA, when first contacted with a protonated cation exchange resin and subsequently contacted with a hydroxylated anion exchange resin, does not bind efficiently or, in some cases detectably, to the latter resin. This observation is unexpected, as numerous publications have detailed methods for purification of DNA based on its efficient binding to anion exchange resins. Since many contaminants of DNA reactions bind as expected to ion exchange resins under these conditions, the result is a simple, rapid and highly automatable method for purification of DNA.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a method for purifying DNA in solution from aqueous samples using porous magnetic particles bearing both positive and negative charges on the surfaces of the particle. It is yet another object of the invention to provide a method for preparing particles having properties that are particularly useful for the purification of DNA by the ion exchange method of the invention. Commercially available ion exchange resins are magnetized in the method of the invention by mixing the resin particles with either uncharged or counter-charged magnetic nanoparticles and separating the unbound nanoparticles in a washing process. The magnetized ion exchange resins are then added either sequentially or simultaneously to the DNA-containing sample using an excess of anionic charge-equivalents in the cation exchange resin. The magnetic resins are then separated from the sample by the use of a magnet, leaving purified DNA.
DETAILED DESCRIPTION OF THE INVENTION
In the method of the invention, mixed-bed ion exchange resins can be used to purify DNA in solution by selectively removing contaminants. The contaminants in question are primarily salts, nucleotides and (in PCR reactions) primers. As these are some of the principal contaminants of enzymatic reactions used in the processing and analysis of DNA, this has considerable commercial utility. Examples of reactions that may be purified using this system include sequencing and PCR reactions, as well as a variety of other enzymatic reactions. When the ion exchange resins are used in a batch format and are present in limiting quantities, binding of salts, nucleotides and primers occurs more efficiently than the binding of the DNA of interest. By manipulating the anion exchange resin and by the addition of linear polyacrylamide, products may be developed that function to selectively separate double stranded DNA from short single stranded DNA, salts and nucleotides or to selectively separate single stranded DNA from salts and fluorescently labeled (and unlabeled) nucleotides. Addition of solid phase reagents useful for protein or detergent removal can be used to further purify DNA samples. An example of such a reagent is SOPE resin (Edge BioSystems, Gaithersburg, MD). Other reagents with similar characteristics may also be used.
Prior to use, both the cation exchange resins and the anion exchange resins used in the method of this invention are prepared by washing commercially available resin suspensions with either water, if the resin is available in a suitable form, or with an appropriate acid or base, acid for a cation exchange resin and base for an anion exchange resin. Examples of resins useful in this invention include, AG® MP-1 (BioRad Laboratories, Redmond, Calif.), Dowex® 2×8 (Sigma, St. Louis, Mo.), Amberlite® IRP-64 (Sigma), CM52 (Whatman), MagaCell-CM (Cortex Biochem, San Diego, Calif.), and Amberlite® CG-50 (Sigma). Many other ion exchange resins are available in a variety of sizes, porosities and functional groups and may be empirically screened for suitability of use in the method of the invention. For the purposes of the invention, the cation exchange resin must be in the protonated form and the anion exchange resin must be in the hydroxylated form. Binding of salts and other contaminants to the resin results in the displacement of either the proton or the hydroxyl group from the respective cation or anion exchange resins. Consequently, water is formed as the sample is purified. For some applications, additional washes with solvents or solutions other than acids and bases may be required to remove soluble interfering substances present in commercially available resins, that interfere with downstream processing. For example, in certain cases anion exchange resins to be used for clean up of sequencing reactions should be washed with polar solvent to remove substances that interfere with chromatographic separation of the product. By way of example, but without limitation, useful polar solvents for this cleanup procedure include methanol, ethanol and isopropanol. The preferred solvent is ethanol. Washing is performed in column or batch formats according to standard methods. The preferred format is the column format. A typical washing procedure for preparation of Dowex 2×8 400 anion exchange resin is described in example 1. This example is provid
Candau Reyes
Kristyanne Eva
Seed John L.
Banner & Witcoff , Ltd.
Edge Biosystems, Inc.
Qian Celine
Yucel Remy
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