Method for extracting DNA from organisms

Abstract

A method for the indirect extraction of DNA greater than 300 kb in size from non-cultivatable organisms contained in an environmental sample is disclosed. The method comprises isolating the organisms from the sample and embedding the isolated organisms in a block of agarose where the organisms are subsequently lysed and the DNA subjected to a first alternating field electrophoresis to extract DNA fragments which are large in size and separate them from the cell debris. The first electrophoretic migration is followed by an enzymatic restriction step and additional electrophoretic migrations. The invention also encompasses DNA obtained by the method.

Description

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 p...

AI Technical Summary

Benefits of technology

The patent text describes a method for extracting DNA from non-cultivatable bacteria in soil and other environments. The method involves chemically and / or enzymatically lysing the bacteria and using a DNA library to capture the DNA fragments. The technique allows for the efficient extraction of DNA fragments greater than 300 kb in size, which are important for new antibiotic and other active metabolite discovery. The method is faster and more efficient than traditional methods and can be used to access the vast potential of non-cultivatable bacteria in soil and other environments.

Problems solved by technology

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.

However, such a technique has the drawback of being relatively slow and tedious.

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.

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.

It results therefrom that the DNA libraries obtained are often highly contaminated with recombinant bacterial clones containing undesired DNA (eukaryotic DNA, degraded extracellular DNA).

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.

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