WO2006016023A1

WO2006016023A1 - Method for extracting dna comprising a enzymatic digestion stage

Info

Publication number: WO2006016023A1
Application number: PCT/FR2005/001511
Authority: WO
Inventors: Joël FLEURENCE; Yolaine Joubert
Original assignee: Universite De Nantes
Priority date: 2004-06-30
Filing date: 2005-06-16
Publication date: 2006-02-16
Also published as: FR2872513B1; FR2872513A1; CN1993465A; CN1993465B

Abstract

The inventive method for extracting AND of a vegetable matter consists in enzymatically macerating said vegetable matter whose pH ranges from 5 and approximately 7 at a temperature ranging from 35 and 40 °C, in centrifuging the extract of the macerated vegetable matter in such a way that a base containing the DNA of the vegetable matter is obtained, in extracting said DNA of the vegetable matter contained in the base and in purifying and amplifying the DNA.

Description

DNA extraction method including an enzymatic digestion step

The invention relates to methods of extracting DNA from a plant material (and more particularly from algae), the DNA obtained being intended to be amplified by PCR or extracted in order to be exploited in bulk quantities .

The invention is likely to be applied in various industrial sectors, and in particular the cosmetic industry or the biotechnology industry (via the improvement of the Polymerase Chain Reaction (PCR) process applied to DNA extracted from plants and more especially seaweed). In the field of DNA extraction techniques, for purposes of analysis or regeneration of a plant species, it is preferable to work on protoplasts.

The production of these protoplasts involves the enzymatic hydrolysis of the cell wall, subsequently allowing easier removal or labeling of the DNA contained in the protoplast nucleus.

Moreover, in the contexts of analysis and / or regeneration of plant species, it is sought, during the production of protoplasts, to maintain the viability of the cells, especially for the purpose of ultimately producing whole plants. The operating conditions of enzymatic hydrolysis of the wall leading to the production of protoplasts, in particular of algae, are classically the following ones (ex: Araki et al, 1998. Journal of Marine Biotechnology, 6: 193-197): a pre- enzymatic treatment in a medium generally containing a protease (eg Papain), an osmoticum (eg Mannitol) and a MES buffer whose pH is adjusted to 7.5; an enzymatic maceration (or enzymatic hydrolysis of the wall) composed of a mixture of enzymes (macerozyme, cellulases ...), an osmoticum and a MES buffer whose pH is adjusted to 6.5; the optimal incubation conditions are obtained for a duration of 2.5 hours and a temperature of 22 ^° C. However, such operating conditions are clearly intended for experimental applications and for maintaining the viability of cells freed from their walls.

Another extraction technique involves a grinding operation of the plant material and more particularly algal to extract the DNA. This extraction technique is limited by the presence of parietal polyssacharides which decrease the solubility of the extracted DNA and / or prevent a correct fixation of TAQ polymerase for DNA amplification via the PCR technique.

The invention therefore particularly aims to overcome the disadvantages of the prior art.

More specifically, the object of the invention is to propose a DNA extraction process that makes it possible to provide quantities that are suitable on an industrial scale.

The invention also aims to provide such a method that is simple in design and easy to implement.

These objectives, as well as others which will appear later, are achieved thanks to the invention which relates to a process for extracting DNA from a plant material, characterized in that it comprises the steps of:

- enzymatic maceration of said plant material, at a pH of between about 5 and about 7 and at a temperature between about 35 ° C and 40 ⁰ C; centrifuging an extract of said macerated plant material leading to obtaining a pellet containing DNA of said plant material; extraction of said DNA from said plant material contained in said pellet;

a purification step of said DNA;

an amplification step of said DNA.

In this way, the combination of the enzymatic maceration technique and the particular operating conditions makes it possible to envisage the extraction of DNA with a yield significantly greater than the yields of the processes of the prior art.

In fact, the degradation of the cell wall and the bursting thereof caused by the operating conditions makes it possible to extract larger amounts of DNA than with the known methods.

Of course, such a method is part of an approach fundamentally different from the prior art, since it leads to the destruction of cells whereas the production of protoplasms is intended to maintain the viability of the cells. In addition, the enzymatic maceration step avoids the contamination of the DNA by the polysaccharides which are likely to limit the fixation of the polymerase.

The amplification step can therefore be performed with excellent efficiency. Advantageously, said extraction step is carried out by dissolving said pellet in a saline buffer whose phH is approximately 8, and by treatment with phenol / chloroform / isoamyl (25: 24: 1 v / v / v)

Preferably, said purification step is carried out by precipitation with isopropanol. According to an advantageous application, the process is intended for the extraction of algal DNA.

It is noted that a process according to the invention can also be applied to other plant materials, for example terrestrial plants which also contain polysaccharides limiting PCR (Polymerase Chain Reaction). According to a particular first application, the method is intended for the extraction of red algae DNA from the Palmaria species, said enzymatic maceration step being carried out using a solution comprising a mixture of xylanases and cellulases. .

According to a second particular application, the process is intended for the extraction of red algae DNA from the Gracilaria species, said step of enzymatic maceration being carried out using a solution comprising a mixture of glucanases and cellulases.

Of course, by selecting appropriate enzymes, the invention is also applicable to all other algae (macro and microalgae) belonging to the groups of red algae (eg Porphyridium sp), green (ex: Ulva sp, Enteromorpha sp) or brown (ex: Fucus sp, Ascophyllum sp, ...).

According to an advantageous solution, said cellulases are celluclasts. In the case of the first application mentioned above, said xylanases are preferably Shearzym. In the case of the second application mentioned above, said glucanases are preferably Finizym.

According to a preferred embodiment, said enzymatic maceration step is carried out for a period of between about 6 hours and about 12 hours. Such a duration makes it possible to achieve a sufficient breakdown of the cells and a complete or almost complete degradation of the polysaccharides.

Other characteristics and advantages of the invention will emerge more clearly in the light of the following description of two variant embodiments of the invention, given by way of illustrative and nonlimiting examples, with reference to the single figure.

As already mentioned, the principle of the invention consists of combining steps and operating conditions likely to increase the extraction efficiency and that of the DNA amplification. In other words, the process makes it possible: to improve the extraction efficiency of the DNA by enzymatic hydrolysis of the cell wall of the algae;

the improvement of the polymerization reaction of the algal DNA (via PCR) by enzymatic degradation of the polysaccharides contaminating this DNA. The use of the enzymatic maceration process as pretreatment of algal DNA (extraction of DNA, removal of polysaccharides limiting the binding of, for example, TAQ Polymerase), pH conditions

(between 5 and 7) and temperature (between 35 ° C and 40 ⁰ C) are particularly critical for obtaining this result.

The expected applications are for example the use of this method for obtaining algal DNA for cosmetic purposes (eg hydrating active ingredient, etc.) or others.

In the context of the process according to the invention, the following description will be made of the use of enzymes (xylanases, cellulases, or glucanases) which degrade the wall of red algae (Rhodophyceae) belonging to the genera

Palmaria and Gracilaria to improve DNA extraction and amplification by PCR (Polymerase Chain Reaction).

The technical protocol is as follows. The algae belonging to the genera Palmaria or Gracilaria, such as the algae Palmaria palmata (Dulse) or Gracilaria verrucosa (Ogo-nori), are macerated in the presence of an enzymatic solution whose composition for each seaweed is as follows: mixture of glucanases (eg Finizym novo) and cellulases (eg Celluclast novo) for the species Gracilaria verrucosa; a mixture of xylanases (ex: Shearzyme novo) and cellulases (Celluclast novo) for the species Palmaria palmata.

The operating conditions of the maceration (minimum and optimal) applicable to the two algae are described below, the maceration being carried out for a period of time ranging from 6 hours to 12 hours. The amount of enzymes used is a function of the amount by weight of the algae to be treated. It is expressed in units / g of seaweed (U / g). By way of a reference, a unit U corresponds to the amount of enzyme capable of releasing a mole of oste in a minute at a temperature of 40 ^° C. The optimum hydrolysis conditions for each alga are described in the tables below: Palmaria almata

Gracilaria verrucosa

An incubation without enzymes was carried out under the same conditions and served as a control to evaluate the relative efficiency of the enzymatic maceration process. The DNA is assayed by measuring the absorbance at 260 nm. Its degree of purity is evaluated by the ratio: Abs 260 / Abs 280 nm.

Referring to Figure 1, the maceration step (step 1) is followed by a centrifugation step (step 2), set to reach 10,000g to 20,000g.

At the end of the centrifugation step, a supernatant containing, inter alia, oligosaccharides and, on the other hand, a pellet containing the DNA of the macerated plant material which is subjected to a step of extraction (step 3), then a purification step (step 4). The extraction step is carried out by dissolving in a pH 8 saline buffer and by treatment with a phenol / chloroform mixture of the pellet obtained at the end of the centrifugation step, the purification step being carried out by precipitation. with isopropanol.

The following table provides a comparison, in terms of extraction of the DNA of the algae Palmaria palmata, between the conventional method (crushing of the alga in buffered medium) and the process according to the invention (without grinding and incubation in presence of xylanases, in this case Shearzyme, and cellulases, in this case Celluclast).

Palmaria almata

It is therefore noted that, on the alga Palmaria palmata, the method according to the invention allows a 41% recovery of DNA recovery, compared to the conventional method.

The following table provides a comparison in terms of extraction of the DNA of the algae Gracilaria verrucosa, between the conventional method (crushing of the alga in a buffered medium) and the process according to the invention (without grinding and incubation in the presence glucanases, in this case Finizym, and cellulases, in this case Celluclast. Gracilaria verrucosa

It is therefore noted that, on the alga Gracilaria verrucosa, the method according to the invention allows a 20% recovery of DNA recovery compared to the conventional method.

The DNA thus extracted then undergoes an amplification step (step 5 in FIG. 1).

As already mentioned, the enzymatic maceration step allows an amplification of the DNA of Palmaria palmata and Gracilaria verrucosa thanks to the enzymatic hydrolysis of the polysaccharides (xylans or glucans, or agar or cellulose) which contaminate the DNA and prevent the fixation on the latter of the polymerase (ex: TAQ polymerase) used for the amplification.

This result has also been observed for microalgae belonging to the Diatom group and appears to be transposable to all macro and microalgae for which DNA amplification by PCR is limited by the attachment of polysaccharides to this DNA.

Claims

1. A process for extracting DNA from a plant material, characterized in that it comprises the steps of:

- enzymatic maceration of said plant material, at a pH of between about 5 and about 7 and at a temperature between about 35 ° C and 40 ° C;

centrifugation of an extract of said macerated plant material leading to the production of a pellet containing DNA of said plant material; extraction of said DNA from said plant material contained in said pellet;

a purification step;

an amplification step of said DNA.

2. Method according to claim 1, characterized in that said extraction step is carried out by dissolving said pellet in saline buffer whose pH is about 8 and by treatment with chloroform / phenol / isoamyl

3. Method according to one of claims 1 and 2, characterized in that said purification step is carried out by precipitation with isopropanol.

4. Method according to any one of claims 1 to 3, characterized in that it is intended for the extraction of algal DNA.

5. Method according to claim 4, characterized in that it is intended for the extraction of DNA of red algae of the Palmaria species, said enzymatic maceration step being carried out using a solution comprising a mixture of xylanases and cellulases.

6. Method according to claim 4, characterized in that it is intended for the extraction of DNA from red algae of the Gracilaria species, said enzymatic maceration step being carried out using a solution comprising a mixture of glucanases and cellulases.

7. Method according to one of claims 5 and 6, characterized in that said cellulases are celluclast.

8. Method according to one of claims 5 and 7, characterized in that said xylanases are Shearzym.

9. Method according to one of claims 6 and 7, characterized in that said glucanases are finizym.

10. Method according to any one of claims 1 to 9, characterized in that said enzymatic maceration step is carried out for a period of between about 6 hours and about 12 hours.