Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-12-19
2003-01-28
Sisson, B. L (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S287200, C436S094000, C536S023100, C536S024300, C536S024330, C204S456000, C204S461000
Reexamination Certificate
active
06511831
ABSTRACT:
DESCRIPTION
The present invention relates to a procedure and a device for isolating nucleic acids from a sample, in particular a biotic or abiotic material.
The specific or quantitative isolation of nucleic acids from certain source materials plays a great role in a large number of scientific, industrial or other areas. Such areas are for example environmental analysis, forensic technology, basic research, foodstuff diagnosis, veterinary diagnostics, epidemiology, tumour diagnostics, genetic material analysis, analysis of genetic and infectious diseases, monitoring of diseases, therapy of diseases, early detection of multiple-drug resistant germs, soil analysis, research and development of pharmacological substances and vaccines, agro-technology or the like. The source materials for the isolation of the nucleic acids can be as diverse as the application areas, for example eukaryotic or prokaryotic cells or their homogenates, soil samples, blood samples, body fluids or tissue homogenates. Depending on this source or sample material different analysis procedures must be used, for example, in order to make the nucleic acids present in cells and/or cell nuclei accessible for isolation. Treatment by ultrasound and/or enzymes are examples of commonly used dissociation procedures. After carrying out the dissociation treatment, the nucleic acids are isolated for example by way of gel electrophoresis, ultra-centrifugation or affinity chromatography.
In contrast to traditional procedures such as enzyme-immuno-assays (EIA), cell cultures and animal tests, genetic investigations using appropriate nucleic acid diagnostics yield, rather rapidly, more sensitive and more specific results, so that costs can be reduced. Nucleic acids diagnostics is based on the isolation of nucleic acid, that is, the preparation of the sample for releasing nucleic acids from biological material, the purification of nucleic acids, their detection and the subsequent analysis —vide for example Lichtensteiger et al., J. Clin. Microbiol. 34 (1966), 3035 to 3039.
Suitable for deployment in nucleic acid diagnostics, affinity chromatography is based essentially on the capacity of nucleic acids to reversibly bind to positively charged and/or positive-pole matrices. Usually at first an anionic or polar binding of the nucleic acids to the matrix is effected, and subsequently impurities are removed from the nucleic acid by means of suitable solvents. In a second step the nucleic acid bound to the matrix is detached from the matrix by means of a further solvent, for example one having a greater ion density. Subsequently the nucleic acid thus isolated will generally have to be de-ionised so it can be used in further analyses.
It proves disadvantageous in this case that, depending on the sample material and the dissociation procedure used, different strategies for isolation must be developed and deployed. In addition, it is not possible to carry out sample dissociation and nucleic acid isolation respectively purification in a single step.
Both DNA and RNA are commonly also isolated by ultra-centrifugation, in which case protease and phenolic treatments must often be employed. This modus operandi has the disadvantage that in particular DNA of high molecular weight will often remain contaminated with impurities even after the isolation process, and that the molecules are exposed to the danger of breaking up due to the shear forces acting upon them. Furthermore, the phenolic treatment is damaging to health and to the environment.
A further method is based on the binding of nucleic acids to silica materials under high-salt conditions, such as 6M GdnSCN, which would then also have been used earlier as a cell dissociation agent. The use of such materials now proves a disadvantage because of their poisonous nature, viscosity and inhibition effect on subsequent processes such as PCR.
Gel electrophoresis, often used to isolate nucleic acids, also has its disadvantages, among other things due to the required relatively laborious pre- and post-processing of the samples respectively the nucleic acids.
The technical problem on which the present invention is based thus lies in the task of providing a cost-effective and simple procedure for cell dissociation and nucleic acid isolation that permits the. highly specific preparation, from any biotic and/or abiotic sample material, of particularly pure nucleic acids in a single step already during the dissociation of the sample. This procedure is to be capable, in principle, of quantitatively isolating nucleic acids as a class of substances from many single individuals, as well as specifically isolating and purifying quite specific nucleic acid sequences.
The invention solves this problem by providing a procedure for isolating nucleic acids from a sample, with the sample being dissociated under the influence of at least one electric field and the nucleic acids released being brought into contact with a nucleic acid-affine material in such a way that at least part of the nucleic acid binds to the nucleic-acid affine material. The invention in particular relates to an afore-mentioned procedure, with a free respectively uncombined or immobilised nucleic acid-affine material being so brought into contact with the sample, after this has been electrically dissociated, that a combination respectively hybridisation of nucleic acids present in the sample can take place with the nucleic acid-affine material and whereby, as the case may be, the bound nucleic acids can, after a washing step effected as required, be separated from the nuclear acid-affine material and for example be amplified, detected, or otherwise utilised. In this context “affine” can also refer to the. binding of nucleic acids to positively charged materials.
The invention thus provides an affinity chromatography procedure for isolating nucleic acids, whereby the nucleic acids contained in a sample are—after dissociation of the sample by means of at least one electric field—brought into contact with a nucleic acid-affine material and this nucleic acid-affine material binds specific individual, several or all respectively essentially all nucleic acids such as for example DNA or RNA that are contained in the sample and thus isolates them from the other components, of the sample such as proteins, carbohydrates, fats or, as the case may be, nucleic acids that are not of interest. The hybridisation conditions that must be maintained to this end, such as temperature and buffer composition and/or the electrical parameters of the field such as pulse count, pulse frequency or field strength, depend on each concrete isolation task at hand.
The invention thus advantageously provides a single-step procedure, whereby in the course of a single process step a sample is dissociated under the influence of at least one electric field and simultaneously or subsequently the nucleic acids released and originating from the sample are brought into contact with a nucleic acid-affine material in such a way that, depending on the characteristics of the nucleic acid-affine material deployed, either quantitatively all or essentially all nucleic acids or only single nucleic acid groups respectively individual nucleic acid sequences can be separated.
The invention makes possible a universally utilisable nucleic acid diagnostic technology for gene and genome analysis that is capable of being standardised and automated and which has a multitude of application possibilities. The procedure according to the invention is very speedy, with the material effort deployed being low and the use of costly and poisonous substances being minimal. Finally the risk of cross-contamination is reduced, so that the procedure is very sensitive. In addition the procedure can be carried out cost-effectively. The procedure according to the invention furthermore has the advantage of achieving such a high degree of purification of the nucleic acids during nucleic acid isolation and purification that subsequent amplification of nucleic acids is possible without interference from amplification
Bernhagen Jürgen
Brunner Herwig
Elkine Bentsian
Geiger Georg
Tovar G{umlaut over (u )}nter
Fraunhofer-Gesellschaft zur Forderung der Angewandten Forschung
Ostrolenk Faber Gerb & Soffen, LLP
Sisson B. L
LandOfFree
Electrical integrated nucleic acid isolation, purification... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Electrical integrated nucleic acid isolation, purification..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Electrical integrated nucleic acid isolation, purification... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3001305