Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-08-08
2001-01-30
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S025310, C536S025410
Reexamination Certificate
active
06180778
ABSTRACT:
The present invention pertains to a process for the chromatographic separation of nucleic acid mixtures into their double-stranded and single-stranded nucleic acid fractions by simultaneously absorbing whole nucleic acids to a mineral support, followed by separation into double-stranded nucleic acid and single-stranded nucleic acid by fractional elution, or by selectively absorbing double-stranded nucleic acid or single-stranded nucleic acid of a liquid sample to a mineral support, as well as solutions and a kit for performing the process according to the invention.
The preparation of nucleic acids, both RNA and DNA, has increasingly gained importance. This involves, for example, lysing the biological sources from which the RNA or DNA is to be isolated, for instance, by mechanical action or chemical action, such as treatment with detergents etc. Thus, the cell lysis for recovery of the nucleic acid is usually followed by a cesium chloride density gradient centrifugation or an extraction with phenol. Although useful for the isolation of nucleic acids, these methods have drawbacks which make their use difficult. Thus, cesium chloride density gradient centrifugation requires the use of time-consuming and expensive ultracentrifugation while working with phenol is questionable for workers' protection reasons.
Thus, attempts to simplify the isolation of nucleic acids have been abundant in the past.
DE 36 39 949 A1, DE 40 34 036 A1 or DE 41 39 664 A1 are concerned, e.g., with improvements of nucleic acid purification by chromatographic methods while avoiding methods which require much equipment, such as high pressure liquid chromatography (HPLC). Although these methods already represent a progress over, for example, ultracentrifugation or phenol extraction, they are relatively complicated technically and labor-intensive. Since a number of successive purification steps is frequently required for fractionation, processing of small sample quantities is particularly problematic, e.g., due to substance losses.
EP 0 389 063 A2 also pertains to a process for the isolation of nucleic acids. The source containing the nucleic acids is lysed in the presence of chaotropic ions and then treated with a material which will adsorb nucleic acids under such conditions. As such materials, there are mentioned diatomaceous earth or other silica-containing mineral supports. It is possible according to the method mentioned in EP 0 389 063 A2 to simultaneously isolate RNA and DNA, and RNA and ssRNA. A desirable fractionation of the nucleic acids bound to the silicon dioxide support into DNA and RNA fractions is not achieved, however. RNA can then be digested by the addition of RNase, leaving the DNA.
In U.S. Pat. No. 5,155,018, Gillespie et al. disclose a process for the isolation and purification of biologically active RNA from biological sources containing RNA, DNA and other cell contents. The source containing RNA is contacted with particles which consist of silica gel containing materials, such as finely divided glass. The binding buffer from which the RNA is adsorbed to the material is acidified solutions containing chaotropic salts. Under such conditions, RNA is bound to the silica material while DNA is not. The use of acidified chaotropic buffers has the drawback that an acidification of binding buffers containing guanidinium thiocyanate (GTC) involves the risk of hydrogen cyanide formation, and thus particular precautions must be taken. Also, the DNA is destroyed by the action of acid. In addition, DNA purification from the authentic sample cannot be performed by this method.
In U.S. Pat. No. 5,075,430, Little describes a process for the purification of plasmid and other DNA, both single-stranded and double-stranded, by immobilizing the DNA on diatomaceous earth in the presence of a chaotropic agent, followed by elution of the DNA with water or a buffer of low salt content. Purification of DNA/RNA is not possible according to this method.
In “Analytical Biochemistry” 121, pages 382 to 387 (1982), M. A. Marko et al. describe a process for the isolation of highly purified plasmid DNA on a large scale using alkaline extraction and binding to glass powder. A fractionation and separate purification of RNA and DNA from a single sample is not described.
The raw preparation of the nucleic acids is followed by subsequent reactions. These subsequent reactions make certain demands on both the isolation procedure and the purity and integrity of the isolated nucleic acids. Especially when followed by enzymatic amplification reactions, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), NASBA (nucleic acid sequence-based amplification), or 3SR (self-sustained sequence replication), the preparation of the nucleic acids should be possible without the risk of cross-contaminations by other samples, and the isolated nucleic acids should be free of interfering cell components and/or metabolites. Due to its specificity and sensitivity, enzymatic amplification of DNA (e.g. PCR) or RNA (e.g. RNA-PCR) is gaining importance, not only in basic research, but also increasingly in the medical field for diagnostic use, such as, for example, for the detection of nucleic acid sequences from minute amounts of cells and/or tissues or biopsy materials, or for the detection of viral nucleic acids from blood or plasma. In addition to the requirements mentioned, these applications make the highest demands on yields and reproducibility of the process for the isolation of nucleic acids.
One object of the invention is to provide a process which is successful not only in separately purifying RNA and DNA from the same biological sample, such as cell lysates and tissue lysates, but generally in separating double-stranded from single-stranded nucleic acids. Operation of the process should be as inexpensive as possible, for example, by using inexpensive unmodified separating materials. In addition, the process should also be suited for sample preparation for diagnostics and be compatible with various amplification methods. Further, the drawbacks mentioned in the discussion of the prior art should be avoided.
Surprisingly, the object of the invention is achieved by a process having the features of four process alternatives; to solutions for use in the process according to the invention or the use of such solutions, and to a kit containing the components necessary for performing the process according to the invention.
In more detail, the process according to the invention for the fractionation of double-stranded and single-stranded nucleic acid structures from biological sources is represented by the following process alternatives.
The sample containing the nucleic acid types to be separated (single-stranded and double-stranded ones) is treated with at least one mineral support wherein the treatment conditions are adjusted with an appropriate aqueous mixture of salts, especially chaotropic substances, and materials containing alcohol groups, such that the single-stranded nucleic acid fraction is predominantly adsorbed on a first mineral support whereas the double-stranded nucleic acid is not adsorbed. Then, the double-stranded nucleic acid flowing out can be further processed with per se known methods. After optionally performed washing steps, the single-stranded nucleic acid adsorbed on the first mineral support is eluted under conditions of low ionic strength or with water. The non-adsorbed double-stranded nucleic acid collected can be further purified, e.g., by subsequently adjusting the fraction with an appropriate aqueous mixture of salts, especially chaotropic substances, and materials containing alcohol groups to such conditions that the double-stranded nucleic acid becomes adsorbable to a second mineral support and, after optionally performed washing steps, becomes elutable under conditions of low ionic strength or with water.
In a second embodiment of the process according to the invention, the treatment conditions for the separation of single-stranded nucleic acid and double-stranded nucleic acid are adjusted suc
Bastian Helge
Colpan Metin
Feuser Petra
Gauch Simone
Jacobson Price Holman & Stern PLLC
Qiagen GmbH
Wilson James O.
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