Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
1996-03-15
2002-05-07
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S198200, C536S025400
Reexamination Certificate
active
06383393
ABSTRACT:
The object of the present invention is a method for the purification and separation of nucleic acid mixtures by chromatography, the use of said method for purifying nucleic acid fragments that have been subjected to modification reactions, a device for performing said method, an aqueous solution which can be used in the method according to the invention, and the use of said solution.
Adsorbing nucleic acids on glass or silica-gel particles in the presence of chaotropic salts is well-known (Vogelstein, B. and Gillespie, D.(1979); Preparative and analytical purification of DNA from agarose, Proc. Natl. Acad. Sci. USA 76: 615-619). According to this method, using high concentrations of chaotropic salts, such as sodium iodide, sodium perchlorate, or guanidine thiocyanate, DNA is isolated and purified from agarose gels and RNA and DNA preparations are isolated and purified from various extracts (Boom, R. et al. (1990); Rapid and simple method for purification of nucleic acids, J. Clin. Microbiol. 28, 495-503, and Yamado, O. et al. (1990); A new method for extracting DNA or RNA for polymerase chain reaction, J. Virol. Methods 27, 203-210). Although the physical processes resulting in an adsorption of the nucleic acids on mineral substrates in the presence of chaotropic reagents are not understood in detail, it is believed that the reason of this adsorption dwells in disturbances of higher-order structures of the aqueous medium. This leads to adsorption or denaturation of the dissolved nucleic acid on the surface of the glass or silica-gel particles. In the presence of high concentrations of chaotropic salts, this adsorption will occur almost quantitatively. Elution of the adsorbed nucleic acids is performed in the presence of buffers having low ionic strengths (salt concentrations). The prior art methods allow for the treatment of nucleic acids and fragments ranging in size from 100 base pairs (bp) to 50,000 base pairs (bp). To date, it has not been possible, however, to quantitatively separate short single-stranded or double-stranded nucleic acid fragments (100 bp and smaller) from very short (20 to 40 nucleotides) single-stranded oligonucleotides (e.g. primers).
Typically, such nucleic acid mixtures are formed as amplification products, for instance by polymerase chain reaction (PCR). In many cases, the products resulting from this reaction are subsequently analyzed in terms of molecular biology, using the conventional techniques such as DNA sequencing, DNA hybridization, cloning, restriction enzyme analysis, and transformation. Thereby, analytical parameters, such as information about genetic mutations for genetic advising or detection of pathogens in medical diagnostics (e.g. HIV), can be obtained. To be capable of making full use of the potential of those diagnostic methods, quantitative separation or purification of these DNA fragments which are often quite small (100 base pairs) is very important.
Presently available purification methods are based on ultrafiltration, high pressure liquid chromatography (HPLC), or extraction of nucleic acid fragments from agarose gels in the presence of chaotropic salts by precipitaion onto glass or silica-gel particles. For the separation of nucleic acid mixtures comprising for example a double-stranded DNA fragment (100 bp) and a smaller single-stranded oligonucleotide (for instance 39-mer), however, these methods are useful only with low efficiency.
The technical problem of the present invention consists in providing a method allowing to avoid the above-mentioned drawbacks of the prior art. This problem is solved by a method for the purification and separation of nucleic acid mixtures by chromatography according to the features described herein.
An embodiment of the invention pertains to the use of the method according to the invention for the purification of nucleic acid fragments following modification reactions, a device for performing the method according to the invention, and an aqueous solution that can be used in the method according to the invention, and a combination of the device and the aqueous solution.
The method according to the invention makes use of the per se known property of nucleic acids to precipitate onto mineral substrates in the presence of chaotropic salts, solutions of salts having high ionic strengths (high concentrations), reagents such as e.g. urea, or mixtures of such substances and to be eluted by the action of solutions of low ionic strengths (salt concentrations). Thus, Applicant's PCT/EP 92/02775 suggests to first adsorb a nucleic acid mixture contained in a medium of low ionic strength on an anion-exchanging material, to subsequently desorb the nucleic acid by means of a buffer of higher ionic strength and then to adsorb the nucleic acids contained in the buffer of this higher ionic strength on a mineral substrate material in the presence of lower alcohols and/or polyethylene glycol and/or organic acids, such as trichloroacetic acid (TCA). Thereafter, the nucleic acids are eluted preferably by means of water or a buffer solution of low ionic strength.
It has now been found that for the separation of nucleic acids, preliminary purification on anion-exchanging materials can be dispensed with. Surprisingly, excellent fractioning of a nucleic acid mixture can also be accomplished by adsorbing the nucleic acids in the presence of high concentrations of chaotropic salts and desorbing the nucleic acids by means of solutions of low ionic strengths.
Thus, the method according to the invention allows for efficiently obtaining nucleic acid fractions of interest in one operation step without preliminary purification steps by adsorbing the nucleic acids to be separated and eluting them.
If samples containing nucleic acids are to serve as sources of the nucleic acids to be purified and isolated, those sources are digested in a per se known manner, for example by treatment with detergents or by mechanical action, such as ultrasonic waves or disintegration. The solution used to receive the nucleic acids may already contain high concentrations of chaotropic salts. After larger cell constituents that may be present have been removed by centrifugation or filtration (WO 93/11218 and WO 93/11211), the solution is contacted with a mineral substrate material in order to adsorb the nucleic acids from the solution having high ionic strength of chaotropic salts on the mineral substrate.
A modification of the method according to the invention consists in performing digestion of the nucleic acids directly within the buffer system employed for the adsorption. In this case, a particularly favorable nucleic acid distribution can be obtained.
Nucleic acids are commonly obtained from eukaryotic and/or prokaryotic cells (including protozoans and fungi) and/or from viruses. For example, the cells and/or viruses are digested under highly denaturing and, if appropriate, reducing conditions (Maniatis, T., Fritsch, E. F., and Sambrook, S., 1982, Molecular Cloning Laboratory Manual, Cold Spring Harbor University Press, Cold Spring Harbor).
One particular embodiment of the invention is especially useful for the isolation of plasmid or cosmid DNA from
E. coli
. Following lysis of the
E. coli
cells with sodium hydroxide/SDS, the solution is neutralized with potassium acetate (KAc, 0.2-0.9 M).
Normally, the cell lysate is neutralized after SDS lysis by means of 3 M potassium acetate. Then, in order to centrifuge off the cell fragments, 5 M guanidine hydrochloride or another high concentration solution of chaotropic salt is added to the cell lysate. With
E. coli
minipreparations, this will yield about 2-3 ml of the sample to be adsorbed on silica gel which is unfavorable, however, since it will have to be centrifuged off then which takes several hours.
After lysis with sodium hydroxide/SDS, the method according to the invention uses e.g. solutions of salts preferably containing
0.2 M KAc
/ 5.5 M GuHCl,
0.2 M KAc
/ 5.5 M GITC,
0.2 M NaAc
/ 6 M NaClO
4,
0.2 M NaAc
/ 6 M GuHCl, or
0.2 M NH
4
Ac
/ 6 M NaClO
4
.
Thereby, neutralization of the c
Colpan Metin
Feuser Petra
Herrmann Ralf
Schorr Joachim
Qiagen GmbH
Therkorn Ernest G.
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