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Method for extracting dna from organismsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, N-glycoside, , Nucleotide, Polynucleotide (e.g., Nucleic Acid, Oligonucleotide, Etc.)Method for extracting dna from organisms description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060188967, Method for extracting dna from organisms. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of PCT Application PCT/FR01/00992 filed Apr. 3, 2001. PCT/FR01/00992 claimed the priority of French patent application 00.05274 filed Apr. 26, 2000, the entire disclosures of both are hereby incorporated by reference into the subject application. FIELD OF THE INVENTION [0002] The invention relates to a method for indirectly extracting DNA, in particular DNA fragments greater than 300 kB in size, of non-cultivatable organisms contained in an environmental sample. It also relates to the DNA, in particular that greater than 300 kB in size, which can be obtained by the method, and also to a DNA library consisting of the DNA fragments extracted by said method. BACKGROUND OF THE INVENTION [0003] In the context of the search for active molecules, and in particular for new antibiotics, amino acids, enzymes and vitamins, various methods are proposed, the principle of which is based on screening the metabolites produced by organisms originating from various environments. Thus, highly important products with antibiotic, anticancer or pesticidal activities have been isolated from microorganisms such as, for example, Streptomyces, Nocardia or Actinoplanes, etc. [0004] Soil and sediments constitute major environments for searching for new active metabolites, not only by virtue of the very large amount of microorganisms which they contain, but also due to the considerable diversity of these microorganisms. It is known that the soil can contain from 10.sup.9 to 10.sup.11 bacteria per gram, of which a maximum of 1% are cultivatable on synthetic media (see R. I. AMANN, W. Ludwig, K H Schleifer (1995) "Phylogenetic identification and in situ detection of individual microbial cells without cultivate" Microbiological Review vol. 59 No. 11 143-169; W B Whitman, D C Coleman, W J Wiebe, 1998. Prokaryotes: The unseen majority, Proceeding National Academy of Science USA, Vol. 95: 6578-6583). [0005] Moreover, the number of bacterial species per gram of soil is evaluated at 1,000 to 10,000. In fact, with DNA-DNA reassociation techniques, the results indicate that the genetic diversity of the microorganisms is certainly greater than 4 000 species per soil sample (Torsvik et al., 1990, Applied Environmental Microbiology, 56: 782-787). Still based on methods which do not involve culturing microorganisms in vitro, V. Torsvik, J. Goksoyr, F L. Daae, R. Sorheim, J. Michalsen and K. Salte, 1994, p.39-48, in Beyond the Biomass, K. Ritz, J. Dighton and K. E. Giller (eds.), John Wiley and Sons, Chichester, indicate that the diversity of bacterial species may reach 13 000 species per 100 g of soil. Thus, estimating there to be 10.sup.10 bacterial cells per gram of soil, a bacterial species should be represented by an average of 10.sup.6 cells, even for the rarest species. [0006] Moreover, bacteria have been detected in a large number of environments ranging from the stratosphere to the very depths of abysses, including the most extreme media in terms of physicochemical conditions. Thus, bacterial diversity is explained by a diversity of location of bacterial populations, first of all in terms of the macroenvironments which they have colonized, but also in terms of the microenvironments which characterize, for example, and in a nonlimiting manner, the structuring of soils, as described by L. Ranjar, F. Poly, J. Combrisson, A. Richaume, S. Nazaret, 1998. A single procedure to recover DNA from the surface or inside aggregates and in various fractions of soil suitable for PCR-based assays of bacterial communities. European Journal of Soil Biology, 34(2), 89-97. [0007] A large number of studies relating to bacterial diversity, based on the analysis of 16S rDNA ribosomal DNA genes, reveal the presence of many new phyla belonging to the domains of Archaebacteria, but also bacteria. The number of bacterial divisions identified has tripled in 10 years. Thus, the vast diversity of soil bacteria is still unknown. It is largely ignored in scientific research study and is not accessible and therefore not exploited industrially. [0008] One of the objectives pursued is to gain access to this vast potential by extracting the DNA from the 99% of remaining non-cultivatable bacteria. [0009] To do this, a first solution consists in improving culture media by increasing knowledge regarding the physiology of bacteria so as to make them cultivatable and therefore decrease the number of non-cultivatable bacteria. Such a technique has been used since the beginnings of microbiology and has produced very good results. In fact, close to 5,000 microorganisms have been described, all environments taken into account. Thus, more than 40,000 molecules have been characterized and close to half have biological activity. However, such a technique has the drawback of being relatively slow and tedious. [0010] Another solution consists in directly extracting the DNA of non-cultivatable organisms from various environments by chemical and/or enzymatic lysis, as described, for example, in documents U.S. Pat. No. 5,824,485 or WO 97/12991, or else Antonia Suau et al., "Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 65, No. 11, November 1999, pages 4799 and 4807, and P. S. ROCHELLE et al., "A simple technique for electroelution of DNA from environmental samples" BIOTECHNIQUES, Vol. 11, No. 6, 1991, pages 724, 726-728. The environments considered are either of the aquatic type, which requires concentration of the bacterial cells, as illustrated by the works of J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya and E. deLong, 1996--Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon, Journal of Bacteriology, 178: 591-599, or of the terrestrial type. [0011] One of the main drawbacks of this direct extraction technique is that it leads to the extraction of only DNA which is small in size, of the order of a maximum of 1 to 23 kB. In fact, the physicochemical constraints which are imposed on the DNA during this type of experiment lead to its degradation. A. Frostegard, S. Courtois, V. Ramisse, S. Clerc, D. Bernillon, F. Le Gall, P. Jeannin, X. Nesme, P. Simonet, 1999. Quantification of bias related to the extraction of DNA directly from soils. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, VOL. 65 (12): 5409-5420, clearly show that the improvement of DNA extraction yields, in particular by grinding or sonication techniques, leads to great degradation of the DNA recovered. [0012] In addition, the extracted DNA comprises the extracellular DNA contained in the soil, but also the eukaryotic (fungi, plant cells, animal cells) and prokaryotic (bacteria) DNA and the other organisms present in the soil (protozoa, etc.). It results therefrom that the DNA libraries obtained are often highly contaminated with recombinant bacterial clones containing undesired DNA (eukaryotic DNA, degraded extracellular DNA). Moreover, the libraries thus constituted are characterized by DNA inserts which are small in size (less than 30 Kb). These libraries are not therefore suitable for applications such as the analysis of complete genomes or metagenomes, or the search for, study and exploitation of complete or virtually complete metabolic pathways. [0013] Due to the dilution of the target DNAs by the non-target DNAs, it is consequently essential to selectively amplify the target DNA proportion by PCR so as to constitute appropriate DNA libraries, as described in the abovementioned Suau document. This approach has the main constraint of being able to access only genetic information similar to that already known, in fact excluding access to completely new and original DNA sequences. However, the use of defined PCR primers in very conserved regions has made it possible to isolate genes of interest belonging to nonisolated bacteria, as described, for example, by K. Seow, G. Meurer, M. Gerlitz, E. Wendt-Pienkowski, C. R. Hutchinson and J. Davies, 1997--A study of iterative type II Polyketide Synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms, Journal of Bacteriology, 179: 7360-7368. [0014] Other selection methods have been developed. These methods consist in subjecting the bacterial community to a selection pressure making it possible to enrich given bacterial populations. It is thus hoped to have preferential access to the genetic information desired. Other selections based on the DNA composition (% GC) or its complexity have also been described, such as, for example, document WO 99/45154. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIG. 1 represents the migration profile of the bacterial DNA extracted according to example 1 (lane 2), according to example 2 (lane 3) and according to example 3 (lane 4). [0016] FIG. 2 represents the migration profile of bacterial DNA extracted according to example 2, at the end of the second migration (2A) and of the third migration (2B). DETAILED DESCRIPTION [0017] All patents applications, publications, or other references that are listed herein are hereby incorporated by reference. [0018] The problem that the invention proposes to solve is therefore to develop a simple method for extracting, purifying, separating and extracting DNA fragments which are large in size, in particular greater than 300 kB, of non-cultivatable organisms and mainly bacteria. The obtaining of these DNAs which are large in size, purified and extracted in this way, is an indispensable condition for producing genomic and metagenomic DNA libraries which are adequate in the field of application. [0019] Another objective of the invention is to provide a method which makes it possible to purify, separate and extract the target DNA free of any DNA contamination, in particular eukaryotic DNA or degraded extracellular DNA, for the purpose of preparing adequate libraries. Continue reading about Method for extracting dna from organisms... Full patent description for Method for extracting dna from organisms Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for extracting dna from organisms patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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