Method for extracting DNA from organisms

a technology of organisms and extracting dna, which is applied in the field of extracting dna from organisms, can solve the problems of not being exploited industrially, not being accessible, and the vast diversity of soil bacteria is still unknown, and the technique has the drawback of being relatively slow and tedious

Inactive Publication Date: 2006-08-24
NALIN RENAUD +2
View PDF3 Cites 25 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for extracting DNA from organisms
  • Method for extracting DNA from organisms
  • Method for extracting DNA from organisms

Examples

Experimental program
Comparison scheme
Effect test

example 2

EXTRACTION OF THE BACTERIAL FLORA FROM SOIL

[0137] I—Starting Material

[0138] 7.5 g of soil are taken up in 50 ml of a buffer solution comprising 30 mM sucrose, 25 mM EDTA and 50 mM TES.

[0139] II—Grinding of Material

[0140] a) the material is ground in a grinding bowl using a WaringBlender for 1 minute at maximum power;

[0141] b) the ground material is taken up in a 50 ml Falcon tube and centrifuged at 6 000 rpm at 10° C. for 10 minutes;

[0142] c) the supernatant is removed and the pellet is taken up with 50 ml of 0.8% NaCl;

[0143] d) 2 rounds of grinding for 1 minute with a WaringBlender at maximum power are carried out, with a 5 minute pause between the two in ice (cooling of the ground soil material);

[0144] e) the ground material is taken up in a 50 ml Falcon tube and kept in ice.

[0145] III—Carrying Out the Density Cushion Sedimentation

[0146] A 60% weight / volume solution of NYCODENZ is prepared (30 g in the final volume of 50 ml of ultrapure water), corresponding to a density...

example 1

is repeated

EXAMPLE 3

EXTRACTION OF INTESTINAL BACTERIAL FLORA

[0166] I—Starting Material

[0167] 3 g of stools are taken up in 50 ml of 0.8% NaCl.

[0168] II—Grinding of Material

[0169] The grinding is carried out using a sterile Teflon Potter homogenizer in a borosilicate glass cylinder;

[0170] a) grinding for 2-5 minutes in ice;

[0171] b) should the grinding with the Potter homogenizer not be sufficient, the material is subjected to very rapid grinding in a WaringBlender (5-10 seconds);

[0172] c) the ground material is filtered through sterile gauze.

[0173] III—Carrying Out the Density Cushion Sedimentation

[0174] A 30% weight / volume NYCODENZ solution is prepared (15 grams in a final volume of 50 ml of ultrapure water), corresponding to a density of 1.16. A 10 ml cushion of NYCODENZ is then put in place in the ultraclear tube, using a pipette, under the suspension derived from the grinding. The sedimentation is then accelerated for 30 minutes at a temperature of 4° C. by ultracentri...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): C12P19/34C12N15/10G01N27/447
CPCC12N15/1003G01N27/44773
Inventor NALIN, RENAUDROBE, PATRICKVAN, VAN TRAN
Owner NALIN RENAUD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap