Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-06
2004-03-02
Benzion, Gary (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025410, C536S023100, C435S091300, C435S091320, C436S177000
Reexamination Certificate
active
06699987
ABSTRACT:
Subject of the invention are formulations which do not contain chaotropic components for isolating nucleic acids with binding them to a solid phase, in particular DNA, from optional complex starting materials and quantities which contain a lysis/binding buffer system involving at least one antichaotropic salt component, a solid phase and wash and elution buffers known as such. The lysis/binding buffer system may be present as an aqueous solution or as a solid formulation of reaction vessels ready for use. All carriers applied for isolation by means of chaotropic reagents, preferably glass fiber mats, glass membranes, silicon carriers, ceramics, zeolites or materials possessing negatively functionalised or chemically modified surfaces which may be converted to a negative charge potential, may serve as a solid phase.
Furthermore, the subject of the invention is a method for isolating nucleic acids, in particular DNA, from optional complex starting materials with using the formulations according to the invention which is characterized by the lysis of the starting material, binding of nucleic acids to a carrier, washing of the nucleic acids bound to the carrier and elution of the nucleic acids, with amplifying selected sequence sections subsequently, if necessary, and analyzing the multiplied gene sections in the very same reaction cavity subsequently, if necessary. Fields of application of the methods are all laboratories dealing with DNA isolation such as forensic medicine, food diagnostics, medical diagnostics, molecular biology, biochemistry, genetic engineering and all other adjacent fields.
Given classical conditions, DNA are isolated from cells and tissues by decomposing the starting materials which contain nucleic acids under strongly denaturating and reducing conditions, with partly also using protein-degrading enzymes, purifying the escaping nucleic acid fractions in phenol/chloroform extraction steps and obtaining nucleic acids by means of dialysis or ethanol precipitation from the aqueous phase (Sambrook, J.; Fritsch, E. F.; and Maniatis, T., 1989, CSH, “Molecular Cloning”).
The “classical methods” for isolating nucleic acids from cells and, in particular, from tissues, are very time-consuming (partly more than 48 hours), require a remarkable expenditure on apparatuses and, apart from that, are not implementable under field conditions. In addition, such methods are due to the used chemicals phenol and chloroform, to a not insignificant degree, dangerous to health.
Various alternative methods for isolating nucleic acids from various biological starting materials allow to avoid the expensive and unhealthy phenol/chloroform extraction of nucleic acids and a reduction of the expenditure of time.
All these methods are based on a method for the preparative and analytical purification of DNA fragments from agarose gels developed by Vogelstein and Gillespie (Proc. Natl. Acad. Sc. USA, 1979, 76, 615-619). The method combines the dissolution of agarose containing the DNA bands to be isolated in a saturated solution of a chaotropic salt (NaJ) with binding DNA to glass particles. The DNA fixed on the glass particles is subsequently washed with a wash solution (20 mM of tris HCl [pH 7.2]; 200 mM of NaCl; 2 mM of EDTA; 50% v/v ethanol) and thereupon dissolved from the carrier particles.
Till the present day this method has been a few times modified and is, for the time being, applied for various processes of extraction and purification of nucleic acids of various origin (Marko, M. A.; Chipperfield, R. and Bimboim, H. G.; 1982, Anal. Biochem., 121, 382-387).
In addition, today exists worldwide also a multitude of reagent systems primarily for purifying DNA fragments of agarose gels and for isolating plasmide DNA from bacterial lysates, yet also for isolating nucleic acids with longer chains (genomic DNA, cellular total RNA) from blood, tissue or also cell cultures.
All these commercially available kits are based on the principle of binding nucleic acids to mineral carrier in the presence of solutions of various chaotropic salts, which is sufficiently known, and use suspensions of finely grinded glass powder as carriers (e.g. glass milk, BIO 101, La Jolla, Calif.), diatom earths (company Sigma) or also silica gels (Diagen, Del. 41 39 664 A1).
A method for isolating nucleic acids practicable for a multitude of various uses is represented in U.S. Pat. No. 5.234.809 (Boom). There, a method for isolating nucleic acids from starting materials which contain nucleic acids by incubating the starting materials with a chaotropic buffer and a solid phase binding DNA is described. The chaotropic buffers implement the lysis of the starting materials as well as binding of nucleic acids to the solid phase. The method is well suited for isolating nucleic acids from small quantities of materials, being in practice especially applied in the field of isolating viral nucleic acids.
Specific modifications of these methods relate to the use of novel carriers which show advantages in application as to specific aspects (Invitek GmbH WO-A 95/34569).
Yet, decisive drawbacks of the method of isolating nucleic acids from complex starting materials on the basis of incubating the starting material with a chaotropic buffer and a solid phase consist a.o. in the fact that the decomposition of cells to be brought about by the chaotropic buffers is not applicable to all materials or works only extremely insufficiently and with a high expenditure of time also for bigger quantities of starting materials. Apart from that, mechanic homogenisation methods are required if e.g. DNA has to be isolated from tissue samples. Furthermore, various concentrations of different chaotropic buffers have to be always used for investigating various aspects. Thus, the method is, in no way, universally applicable.
Though problems arising due to a possibly difficult lysis of the starting material may be solved by a number of commercially available products for isolating nucleic acids (especially for isolating genomic DNA from complex starting materials), they have the big drawback that no longer a classical “single tube” method is concerned which characterizes the method according to the U.S. patent as the lysis of the starting material is carried out in a usual buffer including a proteolytic enzyme. The chaotropic ions required for the subsequent binding of nucleic acids e.g. to centrifugation membranes have to be added separately to the lysis batch after completing the lysis. But they cannot form part of the lysis buffer as the protein destroying function of chaotropic salts is known and would, of course, immediately destroy the proteolytic enzyme required for an efficient lysis.
That is why the methods of isolating nucleic acids with using chaotropic salts have gained acceptance worldwide in spite of a number of drawbacks and are used a million times by means of commercially available products. These systems are extremely simple to apply and follow always the principle of lysis of starting materials, the subsequent binding of nucleic acid to the solid phase of a glass or silica membrane which is in a centrifugation column on a carrier suspension, washing of the bound nucleic acids and the subsequent elution of the nucleic acids with a buffer of an insignificant ionic strength.
All these systems are based on binding the nucleic acids to the respective carrier surfaces in the presence of chaotropic salts, i.e. at least one buffer solution contains a chaotropic salt as main component. This refers possibly already to the lysis buffer or in the case of systems including proteolytic enzymes a required binding buffer which is added after completing the lysis of the starting material.
The series of Hofmeister for salting out negatively charged, neutral or basic protein solutions form the basis for chaotropic salts. Thus, chaotropic salts are characterized by denaturing proteins, increasing the solubility of nonpolar substances in water and destroying hydrophobic interactions. According to the state of the art notably these properties,
Bendzko Peter
Hillebrand Timo
Benzion Gary
Chakrabarti Arun Kron
Invitek Gesellschaft fur Biotechnik & Biodesign mbH
Norris & McLaughlin & Marcus
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