ES2362100T3 - METHOD TO PRODUCE HIGHLY SENSITIVE ENDONUCLEASES, NEW ENDONUCLEASE PREPARATIONS AND USES OF THE SAME. - Google Patents

Abstract

A method for producing a specific recombinant endonuclease of mismatches in which said method comprises: - expressing said recombinant endonuclease in cells of a complete host plant or of a separate organ thereof, transiently transformed by agroinfiltration with an Agrobacterium strain containing a expression vector comprising a polynucleotide encoding said endonuclease; - isolating said recombinant endonuclease from said cells of the host plant.

Description

The invention relates to the identification and preparation of specific mismatch endonucleases that have high activity and sensitivity and broad substrate specificity.

At the beginning of the last century, the discovery of the possibility of inducing DNA mutations by radiation or chemical products has brought considerable expectation to understand the function of genes in vivo. Since then, mutagenesis and natural sequence variation have been widely used to identify new functions, genes corresponding to a specific function as well as active sites within a specific protein.

A critical aspect for the implementation of this strategy, particularly in the case of point mutations, is the choice of mutation detection methods that are designed to select large stretches of DNA without reducing the sensitivity or specificity of the diagnosis, while providing information about the location of the mutation. Among the most used tools and methods are those based on the defectively matched DNA that could be created in vitro by denaturation and renaturation of two DNA molecules. Mismatches are detected in these heteroduplex molecules using chemicals such as binders or groove molecules that can specifically cleave the single stranded DNA at the mismatch site. Alternatively, single stranded specific endonucleases have been used to cleave DNA at the mismatch site. Most of the endonucleases described herein now belong to the S1 / P1 nuclease class.

It is known that nucleases such as S1, P1 and the Chinese bean nuclease, belonging to the same family called: "S1 / P1 nuclease family" or "S1 nuclease family" cleave DNA into regions of a single chain. However, these nucleases have an optimal acidic pH in the range of 4.0-5.0.

This is disadvantageous for detecting erroneous pairings, since low pH values favor DNA depurinization and destabilize DNA duplexes, which leads to non-specific degradation of DNA and reduces sensitivity and specificity in detection.

Some years ago, in extracts from various plants, OLEYKOWSKI et al. (Nucleic Acids Res, 26, 4597-4602, 1998) detected an erroneous pairing endonuclease activity that had an optimal neutral pH (approximately pH 8) and that performed a single chain cleavage on the 3 'side of a pairing site wrong. These authors described that this erroneous pairing endonuclease activity was associated with manoxil glycoproteins in extracts of alfalfa, asparagus, celery and tomato sprouts. The enzyme from celery, called CEL I, was purified from the celery stalks by successive stages of precipitation in ammonium sulfate, binding to an agarose-concavalin A column and elution by �alpha�-d + -manose, binding to a phosphocellulose column and elution using a linear gradient of KCI, and fractionation by size exclusion chromatography. The preparation of CEL I obtained in this way contained various bands of 34-39 kDa proteins.

YANG et al. (Biochemistry, 39, 3533-3541, 2000) and PCT application WO 01/62974 describe an improved CEL I purification by using alphamethyl-mannoside in the purification buffers to overcome the aggregation of CEL I with endogenous lecithins. These documents also describe the cloning of the CEL I cDNA. Based on sequence data, CEL I was assigned to a subfamily of the S1 / P1 nuclease family and several possible homologs encoded by the Arabidopsis BFN 1 genes (nucleotide of GenBank AY040016), ZEN 1 of Zinnia (GenBank nucleotide AB003131), and Lily DSA6 (GenBank nucleotide AF 082031).

It has been shown that CEL I endonuclease activity is highly specific for mismatches by base substitution and for erroneous matches resulting from insertion / deletion events and that it is independent of the context of the flanking sequence. Therefore this is useful as a mutation detection reagent in various methods that involve mutational selection. The CEL I mismatch detection system is a simple assay that requires amplification of the target sequence by PCR, denaturation and hybridization to allow heteroduplex formation between the wild type and the mutant allele, enzymatic cleavage of the mismatch and Product analysis by gel electrophoresis. This is advantageous over other known mismatch detection systems, such as denaturing HPLC, due to its specificity and sensitivity for detecting mismatches in large stretches of DNA.

As an example, OLEYKOWSKI et al and YANG et al (in publications cited above) indicate their use to detect sequence modifications in the human BRCA 1 gene and SOKUENKO et al (Nucl. Acids Res., 29 e111, 2001) describe their use to detect mutations and polymorphisms in large regions of genomic DNA. CEL I is also used for high-performance selection in TILLNG (Selection of locally induced genome lesions), in which after chemical mutagenesis the selection is made for point mutations, or for the detection of polymorphisms in natural populations, also called "Ecotilling" (COMAI et al, Plant Journal, 37, 778-786, 2004). This has been used in plants (COLBERT et al., Plant Physiology 126, 480-484, 2001; TILL et al., Genome Research 13, 524-530, 2003; PERRY et al., Plant Phsysiology 131, 866-871, 2003) as well as in animals such as zebrafish (WIENHOLDS et al., Genome res., 13, 2700-2707, 2003). PCT application WO 03/066809 proposes the use of CEL I in a method for reclassifying sequence variations between related polynucleotides called "Genetic Reclassification by Wrong Pairing Resolution" (GRAMMR).

However, CEL I has the disadvantage of having an cleavage efficiency that varies from one mismatch to another: in the case of a DNA loop with a single nucleotide insert, OLEYKOWSKI et al. (Nucleic Acids Res. 1998 Oct 15; 26 (20): 4567-602) indicate that the substrate preference of CEL I is G> A> C> T; in the case of mismatches due to base substitutions, the CEL I substrate preference is C / C ≥ C / A ~ C / T ≥G / G> A / C ~ A / A ~ T / C> T / G ~ G / T ~ G / A ~ A / G> T / T. This efficacy is significant in the wrong pairings C / A, C / C, C / T, G / G. A decrease in activity is observed for others and is almost ineffective in the wrong T / T pairing. This variation in the efficiency of cleavage can result in a lower accuracy in the detection of some mutations when the detection of an allele in a group of DNA is needed.

Another limiting disadvantage of the use of CEL I is the poor performance of the available purification methods. OLEYKOWSKI et al., Starting with 7 kg of celery stalk containing approximately 350 g of protein, obtained 3 ml of CEL I at 0.1 µg / µl; the purification procedure described by YANG et al. and in the PCT document WO 01/62974 5 µg of purified CEL I with a specific activity of 3.1 x 107 of CEL I protein units / mg starting from 105 kg of celery stalk were obtained.

To obtain large amounts of CEL I, it has been proposed to produce it by recombinant DNA technology. PCT application WO 03/066809 proposes a broad list of possibly suitable vectors and host cells that include almost any of the known prokaryotic or eukaryotic expression systems; however, the only expression system actually described in this document is a vector based on tobamovirus. The construction resulting from the cloning in said CEL I cDNA vector fused to a sequence encoding a 6-Histidine tag has been used to infect tobacco plants. Recombinant CEL I was recovered from the intracellular fluid of infected plants and purified by metal affinity chromatography on NTA-nickel resin. PCT WO 03/066809 says nothing about the performance of the purified enzyme. This indicates that its activity in a GRAMMR reaction is similar to that of the purified natural enzyme from celery. One of the disadvantages of this system is that viruses tend to recombine, partially or completely losing the expressed gene. This leads to the production of truncated forms in the addition of full-length CEL I, decreasing the specific activity of the enzyme.

PCT application WO2004 / 035771 refers to a method for the production of CEL I in yeasts. For this purpose, a synthetic gene encoding CEL I was constructed by modifying the natural DNA sequence of CEL I according to the use of codon in yeast. This document indicates that the recombinant CEL I produced by this synthetic gene can recognize all possible mismatch combinations and explains with examples the recognition and cleavage of the A / A mismatch. On the other hand, it does not say anything about the mismatch preference of said recombinant CEL I.

From the foregoing it seems that the methods currently available for the production of recombinant CEL I do not provide any significant improvement over the production of natural CEL I from celery. In addition, they do not address the problem of natural CEL I substrate preference.

In addition, it would be desirable to have other endonucleases that could, such as CEL I, cleave mismatches from single base pairs in heteroduplex DNA templates at neutral pH, but that had a different mismatch preference or preferably split all pairings equally well. wrong. However, so far these endonucleases have not been identified.

The existence of CEL I type endonuclease activities has been described in many plants (see, for example, OLEYKOWSKI et al., 1998 indicated above). However, the enzymes responsible for these activities have not been characterized, their chemical properties, such as substrate preference, have not been studied and their sequences have not been identified. On the other hand, CEL I structural homologues have been identified by computer (YANG et al. Cited above; TILL et al. Nucleic Acids Res., 32, 2632-41, 2004). Three of these (BFN1 from Arabidopsis, ZEN1 from Zinnia and DSA6 from lily) have been described as being involved in plant senescence (PEREZAMADOR et al., Plant Physiol. 122, 169-180 2000). However, they have not been purified and remain biochemically uncharacterized: in particular, their efficacy to recognize erroneous pairings in heteroduplex DNA in vitro has not been tested. From spinach, an endonuclease called SP has been purified which, based on its activity, has been assigned to the S1 / P1 family and has an optimal neutral pH. Like CEL I, this enzyme cleaves mismatches by insertions / deletions and by base substitutions; however, it does not recognize those that contain a guanine residue (OLEYKOWSKI et al. Biochemistry, 38, 2200-5, 1999).

Looking for alternative methods to obtain recombinant CEL I, the authors of the invention have tried to express it in plants by means of transient expression mediated by Agrobacterium.

These have expressed the recombinant CEL I in agroinfiltrated tobacco leaves and purified it from leaf extracts by precipitation with ammonium sulfate. Surprisingly, they have discovered that, they obtained a very high yield of recombinant CEL I with a high specificity and also, that said recombinant CEL I preparation recognizes the erroneous pairings with a broader specificity and a greater sensitivity than in the known CEL I preparations. in the prior art, allowing clear detection even of erroneous pairings, such as T / T, which are considered poorly recognized by CEL I.

In view of these results, the inventors have had the idea of using this method to explore endonucleases, identified by computer as belonging to the S1 / P1 family, by testing their activity in vitro.

Therefore, the present invention provides a simple and rapid method of obtaining large amounts of endonucleases, in particular S1 / P1 nucleases, from a small amount of starting material.

In this document, a specific erroneous pairing endonuclease is defined as an endonuclease that is capable of specifically cleaving all erroneous pairing by base substitution (ie A / A, G / G, C / C, T / T, A / G, A / C, G / T, C / T, G / A, C / A, T / C, T / G), as well as by insertions / deletions of one or more nucleotides.

An object of the present invention is therefore a method for producing a specific recombinant endonuclease of mismatch, wherein said method comprises:

-

 expressing said recombinant endonuclease in cells of a complete host plant or of an organism separated therefrom transiently transformed by agroinfiltration with an Agrobacterium strain containing an expression vector comprising a polynucleotide encoding said endonuclease;

-

 isolating said recombinant endonuclease from said cells of the host plant.

Said plant cells may be part of a cell suspension or a tissue or organic culture. In this case, the enzyme can be collected from the supernatant and / or from cultured cells or tissues or organs. Preferably, they will be part of a complete plant or of a separate organ thereof; In this case, the transient transformation with the Agrobacterium strain will be carried out by agroinfiltration.

Agroinfiltration is a method of transient expression based on the supply of Agrobacteria, which contains a gene of interest, within the intact plant tissue.

A DNA construct comprising a gene of interest is cloned into a binary vector and transferred to a selected Agrobacterium strain and the transformed Agrobacterium is grown to a logarithmic phase or to saturation and collected in the same manner as for a mediated transformation. by conventional Agrobacterium. Classically, agroinfiltration is performed by applying a suspension of Agrobacterium cells transformed by injection into an organ (usually the leaves) of the selected plant using a needleless syringe or by vacuum infiltration for a few minutes. After the release of the vacuum, the organ or the whole plant is placed in a culture chamber. The protein of interest expressed is extracted from the infiltrated organ, usually one to four days after infiltration. Agrofiltration protocols are described in various publications, for example KAPILA et al., (Plant Sci. 122, 101-108, 1997; BENDAHMANE et al., (Plant Cell, 11, 781-792, 1999); SCOFIELD et al. ., (Science., 274, 2063-5, 1996); TANG et al., (Science., 274, 2060-3, 1996); MARILLONET et al., (Proc Natl Acad Sci USA, 101, 9852-7 , 2004); WROBLEWSKI et al., (Plant Biotech. J., 3, pp. 259-273, 2005).

The classic protocols used for transient expression mediated by Agrobacterium, and in particular for agroinfiltration, can be used in the practical embodiment of the present invention. A large selection of Agrobacterium strains, binary vectors and regulatory elements that control the expression of the gene of interest is available and one skilled in the art can select among them the most appropriate, for example according to the host plant that is pretend to use

In the experiments described in the following Examples, the inventors have used a binary vector derived from pBIN19, pBIN61, and a strain of Agrobacterium C58C1 that hosts the hypervirulent plasmid pCH32 and the cDNA of the genomic coding sequences has been expressed under the VMCa 35S promoter. However, other binary vectors, other strains of Agrobacterium and other constitutive or inducible promoters with the same result can be used.

Advantageously, the agroinfiltration will be carried out in the leaves, which can optionally be separated from said plant immediately before or immediately after the infiltration.

Host plants that can be used in the method of the invention include any plant that is compatible with Agrobacterium transformation. Preferred plants include in particular those of the genus Nicotiana, in particular Nicotiana benthamiana and Nicotiana tabacum.

According to a preferred embodiment of the invention, said endonuclease is isolated from an agro-infiltrated plant organ, in particular an agro-infiltrated sheet by a process comprising the following steps:

-

 extracting the cellular content of the agroinfiltered organ that expresses said endonuclease;

-

 add to said extract ammonium sulfate at a final concentration of 30% or more and separate the protein precipitate from the supernatant;

-

 add to said supernatant ammonium sulfate at a final concentration of 80% or more and recover the protein precipitate containing the endonuclease.

Said protein precipitate is resuspended in an appropriate buffer, for example, a buffer comprising Tris HCl (pH 8), PMSF and 10% glycerol. This can be used directly or stored at -80 ° C until use.

Alternatively, the total extract obtained after the first stage indicated above can be used as such, without performing precipitation steps with ammonium sulfate.

Optionally, the endonucleases produced by the method of the invention can be further purified by any appropriate method known therein, such as column affinity purification in which CEL I is labeled with a label having an affinity towards a specific column component. . According to a preferred embodiment, CEL I of the invention is provided with a 6-Histidine tag and is purified by metal affinity chromatography on nickel-NTA.

A method to test whether a candidate endonuclease is a specific pairing endonuclease

wrong includes:

a) producing said candidate endonuclease recombinantly by the method of the invention, as

defined above;

b) test said recombinant endonuclease to determine its ability to degrade DNA

single chain;

c) testing said recombinant endonuclease to determine its ability to cleave a fragment of

Heteroduplex DNA of the assay at a previously defined mismatch site;

d) test said recombinant endonuclease to determine its ability to cleave DNA fragments

heteroduplex carriers of all types of mismatches (i.e., pairings

erroneous by substitution of bases (A / A, G / G, C / C, T / T, A / G, A / C, G / T, C / T, G / A, C / A, T / C, T / G, as well as

mismatches by insertion or deletion).

If an endonuclease passes the tests in stages b), c) and d) (that is, if it can degrade single-stranded DNA, to cleave a fragment of heteroduplex DNA from the assay at a previously defined mismatch site and to cleave fragments of heteroduplex DNA carriers of all types of mismatches) is considered as a specific endonuclease of mismatches.

A method for selecting specific endonucleases from erroneous pairings comprises:

a) produce candidate endonucleases recombinantly by the method of the invention, as

defined above;

b) test said recombinant endonucleases to determine their ability to degrade DNA

single chain;

c) test the recombinant endonucleases capable of degrading single-stranded DNA and test them for

determine its ability to cleave a heteroduplex DNA fragment from the assay at a site of

known and well characterized erroneous pairing;

d) select the recombinant endonucleases capable of cleaving a heteroduplex DNA fragment

of the test at a well-known and well-characterized mismatch site, and test them for

determine its ability to cleave heteroduplex DNA fragments carrying all types of

mismatch

e) select the recombinant endonucleases that pass the tests in stages b), c) and d).

The methods defined above may additionally comprise a step consisting of testing said endonuclease (or endonucleases) to determine its sensitivity by testing its ability to detect a mutant allele in a group of DNA, in the presence of the excess wild type allele, and select the endonucleases that are capable of detecting said mutant allele in the presence of the excess wild type allele at least 9 times (ie, a mutant allele in a group of 10), preferably in the presence of the excess wild type allele at least 14 times, even more preferably in the presence of at least an excess of 19 times and to increase the preference, an excess of 29 times, 39 times, 49 times, 59 times.

Preferably, the tests defined above are carried out in a reaction mixture having a pH of 7 to 8, advantageously 7.4 to 7.8 and containing 5 to 20 mM, advantageously 10 mM MgCl 2. Advantageously, said reaction mixture also comprises 0.5 mM to 2 mM and preferably 1 mM DTT. The inventors have also observed that the addition of PEG-8000 from 2% to 10% (w / v) and in particular 5% of the final reaction mixture 5, increases the overall activity of the endonucleases.

Candidate endonucleases that can be tested by the procedures defined above may be among those of the S1 / P1 family. In the PFAM database (BATEMAN et al., Nucleic Acids Res. 32, D13841, 2004) this family is called PFAM 02265. For example, you can use the HMM profile (Hidden 10 Markov Model) constructed from PFAM 02265 as a probe to explore the available DNA sequence data banks, using the HMMER software to identify candidate endonucleases in different plants.

Alternatively, the InterPro code IPR003154 (corresponding to S1 / P1 endonucleases) can be used for the exploration of database content, for example using the following address:

http://www.ebi.ac.uk/interpro/ISpy?ipr=IPR003154

The exploration of TrembI / Swissprot databases with this code identifies 43 proteins (Table 1).

20 Table 1: scan result of the TrembI / Swissprot database with the code IPR003154.

TrembI or Swissprot access number

Description Sequence length Bit (Blastp)

P24504

Nuclease PA3 (EC 3.1.3.6) 270 127

P24289

Nuclease P1 (EC 3.1.30.1) 270 127

P24021

Nuclease S1 (EC 3.1.30.1) 267 107

Q00235

S1 nuclease precursor. 287 109

Q9P356

Nuclease Le1. 310 111

Q7S8Q5

Hypothetical protein 306 114

Q87OT1

Nuclease likely S1. 324 119

Q8NIH8

Nuclease Le3. 298 129

Q25267

3’-nucleotidase / nuclease. 477

Q9GNZ4

3'nucleotidase / nuclease precursor. 378

Q9NJ13

3’-nucleotidase / nuclease. 377

Q9NJY3

Single chain specific nucleotide. 315

Q86GJ3

Nuclease P4. 316

Q8T4M4

Class I nucleasease 3i6

065424

Alleged bifunctional nuclease 362 217

065425

Alleged bifunctional nuclease 454 164

080326

Endonuclease precursor. 303 480

081656

Protein 6 associated with old age. 298 461

081958

Endonuclease 288 289

Q93WW9

Endonuclease type Si (Fragment). 1361 239

Q9ARD4

Alleged nuclease 289

Q9C9G4

Alleged bifunctional nuclease 290 300

Q9FTRO

Alleged bifunctional nuclease. 310

Q9FTR1

Alleged bifunctional nuclease. 311 180

Q9LGA5

EST D48949 (5i554i). 3081 293

Q9LL59

Wrong pairing endonuclease CEL I. 296 627

Q9SXG1

Nuclease I. 290 291

Q9ZR87

Bifunctional Nuclease 328 308

Q9ZR88

Bifunctional Nuclease (Fragment). 280 305

Q9ZR89

Bfnl bifunctional nuclease. 305 445

Q7XND5

0SJNBbOO34l13.4 protein. 252 277

Q7XPN4

OSJNBaOO6ODO6.10 protein. 290 275

Q8LA68

Endonuclease, supposed. 296 281

Q8LCL6

Alleged bifunctional nuclease 290 299

Q8LDW6

Alleged bifunctional nuclease 294 274

068530

Homol of S1 endonuclease. 309

Q8XRE8

Alleged signal peptide protein. 337

Q989R8

Endonuclease 278

Q7P202

Likely endonuclease 274

Q8F378

Nuclease Yes (EC 3.1.30.1). 306

Q8P5Y5

Endonuclease 270

Q8PHA3

Endonuclease 271

Q9SXA6

Bfnl bifunctional nuclease. 305 448

This analysis is preferably completed by performing a Blast in the databases (blastp or tblastn), using a reference protein sequence (for example the CEL I sequence) and selecting the best hits.

5 The following examples will describe in more detail the trials of five candidate Arabidopsis thaliana endonucleases.

The authors of the present invention discovered that one of these endonucleases, represented in the list of

10 sequences attached under SEQ ID No. 2 (the corresponding DNA sequence is represented under SEQ ID No. 1) and corresponding to BFN1 of Arabidopsis thaliana, has a different specificity and a sensitivity much greater than CEL I.

This discovery of the properties of BFN1 as a specific mismatch endonuclease,

15 which until now was unknown, allows us to propose its use as a mutation detection reagent, to detect erroneous pairings resulting from base substitutions, as well as insertions / deletions of one plus nucleotides.

BFN1, as well as any of the specific mismatch endonucleases that can

Identified according to the method of the invention can be obtained in large quantities by the method of endonuclease production.

The present invention also includes recombinant endonuclease preparations that can be obtained by the production method according to the invention. These are in particular CEL I preparations

Recombinant 25 and recombinant BFN 1 preparations.

The recombinant CEL I preparations of the invention have a different mismatch specificity and greater sensitivity than the prior art CEL I preparations. The recombinant CEL I preparations of the invention have the following mismatch preference: T / G  A / G  G / G  G / T  30 T / T  G / A ≥A / A  C / C ≥T / C  C / T> A / C  C / A, while the preference for mismatch of CEL I separations of the prior art is C / C ≥ C / A  C / T ≥ G / G > A / C  A / A  T / C> T / G  G / T  G / A  A / G> T / T. The recombinant CEL I preparations of the invention can recognize a mutant allele in the presence of

23 times more of the wild-type allele while CEL I preparations of the prior art do not effectively recognize any mutant allele when diluted 8 times in a dilution.

The recombinant BFN 1 preparations of the invention also have a specificity of pairing

5 different from that of the CEL I preparations of the prior art and the recombinant CEL I preparations of the invention. The recombinant BFN 1 preparations of the invention have the following mismatch preferences: G / G  G / A  A / G  G / T  T / G> T / T  A / A  C / C  T / C> C / T  A / C  C / A. The following table 2 summarizes the mismatch preferences for each of these enzymes.

10 Table 2: Comparison of mismatch recognition preferences.

Protein

Recognized more effectively Well recognized Recognized less effectively Very weakly recognized

CEL I of celery (prior art)

C / C C / A C / T G / G A / C A / A T / C G / A T / G G / T A / G T / T

Recombinant CEL I according to the invention *

T / G G / G A / G G / T T / T C / C G / A A / A C / T C / A A / C T / C

BFN 1 (ENDO 1)

G / G A / G T / G G / A G / T A / A C / C T / T T / C C / T A / C C / A *

* = BFN1 (ENDO 1) recognizes these mismatches more effectively than recombinant CEL I. ** N.I .: the calculation of the cleavage efficiency is only semi-quantitative; therefore, slight variations in the classification of erroneous pairings may occur, based on their recognition by endonucleases, depending on the investigator and / or the experiment.

Additionally, the recombinant BFN 1 preparations of the invention have a greater sensitivity both in the CEL I preparations of the prior art and in the recombinant CEL I preparations of the invention. The recombinant BFN 1 preparations of the invention can recognize a mutant allele in the presence of 59 times the wild type allele.

The recombinant BFN 1 or CEL I endonuclease preparations of the invention can be used, as a reagent for mutation detection, in any method that involves scanning for pairings.

20 erroneous as mentioned above. They are particularly advantageous in genotyping, in TILLING, high performance TILLING, Ecotilling, GRAMMR, etc. The methods for using the endonuclease preparations of the invention basically involve the same steps as those of the prior art, that is, PCR amplification of the target sequence, denaturation of the amplification product and hybridization to allow heteroduplex formation between the allele. wild type and the

25 mutant, cleavage of heteroduplex by endonuclease and analysis of cleavage products. Advantageously, when these methods are performed, a mixture of different endonucleases can be used for cleavage of the heteroduplex. Due to their high sensitivity, the endonuclease preparations of the invention also allow high-performance methods to identify mutations in a sample. For example, the preparations of

Endonucleases according to the invention can be used to perform methods such as those described in WO 01/75167, with a much larger number of samples since it is possible to group many samples together for analysis. In accordance with the invention, the endonuclease preparations described above can be used to explore one or more mutations in a target gene, in a large number of samples from an organism or an organism.

The cell line derived therefrom, performing the following steps: a) amplifying said target gene or a part thereof of each individual of said population, b) classifying said amplification products in a two-dimensional or three-dimensional matrix, comprising lines , rows (2-D matrix (two-dimensional)) and columns (3-d matrix (three-dimensional)), c) group said amplification products so that different groups are obtained, presented

Each group a row, a line or a column of said matrix, d) add to each group a reference amplification product obtained from a non-mutated gene and incubate said groups under conditions that allow the formation of heteroduplex, e) incubate each group with an endonuclease preparation according to the invention, and f) detect the presence of heteroduplex in said incubated groups.

Alternatively, in the previous method it can be done by first classifying the samples in the matrix, then grouping them (with the addition of the reference) and performing the amplification, incubation and detection steps.

Depending on the number of samples to be scanned, the matrix can be two- or three-dimensional. For example, if 576 samples are to be scanned, a 24 x 24 matrix can be used. If 13,824 samples are to be scanned, a three-dimensional matrix (24x24x24) should be used more appropriately. Only 72 reactions would be necessary to explore this population and the mutated genes would be individualized by the groups in the column, row and line to which it belongs.

The reference amplification product corresponds to an amplification product obtained from a reference gene (as compared to the mutations being explored), amplified with the same primers as those of the target gene in the population. It is important to add a reference amplification product. In fact, although it is quite unlikely that all samples contain the same exact mutation compared to the reference gene, this will ensure that heteroduplex can be formed if the group contains a target gene that contains a mutation.

In addition, the following additional description will illustrate the present invention, which relates to examples illustrating the preparation and properties of the recombinant CEL-I endonuclease, the test of five candidate Arabidopsis thaliana endonucleases, and the identification of BFN 1, (hereinafter successive in this document also referred to as ENDO 1) as a specific endonuclease of mismatch. It should be understood, however, that these examples are provided solely to illustrate the invention and in no way constitute any limitation thereof.

Legends of the figures Figure 1: detection of point mutation in agarose gel. Heteroduplex (mutant and wild-type DNA) had been incubated with different dilutions of recombinant CEL I (D104 to D1000), or without protein (-). Figure 2: Detection of point mutation in acrylamide gel. WT + Mut: wild-type and mutant DNA had been mixed before PCR, thus generating heteroduplex. WT: only wild-type DNA has been used for PCR, thus generating only homoduplex. Mut: only mutant DNA has been used for PCR, thus generating only homoduplex. D100, D500 and D1000: dilutions of the recombinant CEL I protein. In the upper part of the gel the size of the homoduplex (661 bp) is indicated. The 405 bp row shows the fragment labeled with the FAM fluorochrome. The 256 bp row shows the fragment labeled with the ROX fluorochrome. Figure 3: Detection of point mutation in genomic DNA in agarose gel. PCR products were obtained as described below in Example 4, with primers 4-960 and 4-721 (SEQ ID Nos: 3 and 4). They were then digested with different dilutions of preparation of recombinant CEL I obtained by precipitation with ammonium sulfate (as described in example 2) and analyzed on agarose gel. The size of the PCR products for wild-type and mutant alleles of 1-13 rms, is approximately 500 bp (exactly 481 bp). As a result of the detection of the mutation in the heteroduplex DNA, two bands of approximately 200 and 300 bp were obtained. The two bands can be observed on an agarose gel even at a 1/1000 dilution of the protein produced in tobacco.

1: D100; 2: D500; 3: D1000; 4: without enzyme.

A: Terese; B: rms 1.13; C: T + rms 1,13 Figure 4: Analysis of recombinant Cel I activity in all types of mismatches. A series of mutants based on the Rx gene was created by PCR and heteroduplex were obtained by mixing the corresponding amplification products with these different mutants. For the PCR, fluorophore labeled oligonucleotides IRD700 and IRD800 (MWS®) were used and the mismatch detection was performed in LICOR4300 (LICOR®). The figure shows the channels (A) with IRD dye 700 and (B) with IRD dye 800 of a process. Channel A shows the 204 bp fragment with the 5 'end fragment labeled with IRD-700 dye and channel B shows the 438 bp fragment with the 3' end fragment with the IRD-800 dye. Figure 5: Sensitivity of the recombinant Cel I protein produced in tobacco. 1: Wt pea (wild type); 2: Le1; Wt + Le1; 4 Wt + Le1 + 2ADN; 5 Wt + Le1 + 4DNA; 6WT + Le1 + 6ADN; 7 Wt + Le1 + 8ADN; 8 Wt + Le1 + 10ADN. Figure 6: DNA heteroduplex cleavage at the C-A / T-G mismatch site by five candidate endonucleases. (Ho) = homoduplex of DNA; (Ht) = DNA heteroduplex. Figure 7: Detailed analysis of the specific recognition of all types of mismatch with ENDO1 and ENDO5. The same protocol was used as in Figure 4. Figure 8: Assay to measure the detection sensitivity of ENDO1. As a template, a mixture of mutant (Le1) and wild-type (Torstag) DNA was used to determine the activity of ENDO1, in the following proportions: 1 mutant per 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45 and 60 wild type (from left to right). The last two lines of each panel are homoduplex (only mutant and only wild type). Left panel: IRD700 channel, fragment size detected = 338 bp. Right panel: IRD800 channel, fragment size detected = 300 bp. Figure 9: comparison of detection of erroneous pairing by CelI and ENDO1. Lines 1, 5, 8 and 10 correspond to homoduplex. The fragments resulting from the endonuclease cleavage are 405 bp (marked in blue) and 256 bp (marked in red, less visible in black and white). ENDO1 recognizes erroneous pairings more effectively than CelI. In addition, the fund (non-specific activity) is much smaller with ENDO1. Figure 10: Detection of a known point mutation in acrylamide gel with ENDO1 in two different dilutions (D1000 and D5000). M5 and M12 are two plasmids that contain the Rx gene; one contains the Wt form (wild type) and the other the mutated form. Primers: Rx21 rox and Rx22 fam (in this figure, the smallest cleavage band, marked in red, clearly appeared on the gel but is less visible in black and white.

EXAMPLE 1: CLONING, PURIFICATION AND PRODUCTION OF ENDONUCLEASE CEL I 5 CEL I cDNA cloning

Celery RNA Extraction

Using a pestle and a mortar, young leaf tissues (1 g) were crushed in liquid nitrogen. The powder was suspended in 10 ml of Trizol extraction buffer (Gibco). The suspension was mixed with 2 ml of chloroform and centrifuged at 12000 rpm at 4 ° C for 15 minutes. The supernatant was mixed with an equal volume of isopropanol and the total RNA was precipitated by centrifugation at 12000 rpm for 10 minutes at 4 ° C. The sediment was washed with 80% ethanol, dried in air and resuspended in 200 µl of water with DEPC (diethyl pyrcarbonate).

15 DNase Treatment

DNase treatment was performed to hydrolyze the DNA that contaminates the RNA preparation. Following the manufacturer's instructions (Promega), 10 µg of total RNA was incubated with 10 units of DNase. The reaction was incubated at room temperature for 15 minutes then stopped by the addition of EDTA at a final concentration of 25mM and by thermo inactivation of DNase at 65 ° C for 10 minutes.

Synthesis of CEL I cDNA

Ten microliters of DNase treated RNA were used for the synthesis of the first cDNA chain. The

Synthesis of the first cDNA chain was primed with 2 picomoles of 20-unit oligo dT primer. The 50 µl reaction mixture (10 µl of 5x Superscript buffer [GIBCO-BRL], 5 µl of 100 mm DTT, 5 µl of 5mM dNTP) was heated at 70 ° C for 10 minutes and then cooled on ice. 1 µl of Superscript reverse transcriptase (200 µl units; GIBCO-BRL) and RNase 1 µl inhibitor (37 µl units; Pharmacia) were added and the reaction was incubated at 42 ° C for 1 hour. PCR amplification was used to convert the first cDNA chain into double cDNA chain by PCR amplification using two specific primers in the 5 ’and 3’ CEL I (see Table 3 below).

CEL I cloning and expression on tobacco leaves

35 The full-length CEL I open reading phase was amplified by PCR and inserted between the 35S promoter and the VMCa transcriptional terminator in the binary vector pBin 19 to create pBIN35S-CELI. Another construction pBIN35S-CELI8His was also built. The pBIN35S-CELI8His construct is identical to pBIN35S-CELI except that an 8-amino acid Histidine tag was inserted into the C-terminal of the CEL I protein. In the following table 3, the oligonucleotides used to create pBIN35S-CELI and pBIN35S are indicated -CELI8His. TABLE 3

Primer Name

N CEL terminal

5 ’TATCGTTCTAGAGGGAATGACGCGATTATATTCTGTGTTC 3’ SEQ ID No. 5

C CEL terminal

5 ’TATCTGAATTCATGCCAAAGAATGATC 3’ SEQ ID N ° 6

C terminal His 8 of CEL

5’AATTCAATGGTGATGGTGGTGATGGTGATGTGCCAAAGAATGATCTGCGG 3 ’SEQ ID No. 7

These constructs were transformed into the C58C1 strain of Agrobacterium carrier of the virulence auxiliary plasmid pCH32 (Hamilton et al PNAS, 93 (18): 9975-9979, 1996). Plasmid pCH32 expresses VirG and VirE and was used to enhance T-DNA transfer. Agrobacterium cells were inoculated in 5ml of medium with L broth

45 (Sambrook et al. 1989) supplemented with 50 µg / ml kanamycin and 50 µg / ml tetracycline and were grown at 28 ° C overnight. Then, the medium with L broth (50ml) supplemented with 50 µg / ml kanamycin and 5 µg / ml tetracycline was inoculated with the 5ml cultures overnight and grown at 29 ° C for two days. The cells were precipitated and resuspended to a final concentration of DO600 of 0.5 in a solution containing 10 mM MgCl2, 10 mM MES, pH 5.6 and 150 µM acetosyringone. The cultures were incubated at room temperature for two hours before agrofiltration in Nicotiana bentamiana leaves.

The Agrobacterium suspension was injected into the leaves using a needleless syringe.

Agro-infiltrated Nicotiana bentamiana plants were incubated for at least 48 hours at 24 ° C, 16 hours after

55 light, 60% humidity. To test the effectiveness of agroinfiltration, the plants were also agro-infiltrated with a construct that expressed the green fluorescent protein (GFP). Before collecting the leaves, the intensity of the GFP expression for each sheet was monitored using a UV lamp. The leaves of the plant were collected only if the GFP was expressed.

Preparation of tobacco leaf protein by precipitation with ammonium sulfate Agro-infiltrated tobacco leaves were collected and weighed. Two grams of agro-infiltrated sheets were homogenized in 7 ml of buffer containing 0.1M Tris-HCl, pH 8, 200µM PMSF, 0.125nM β-mercaptoethanol and 10% glycerol and then centrifuged at 3000 g for 25 minutes to sediment the cell debris . To the supernatant was added 100% ammonium sulfate to the final concentration of 30% and then the samples were incubated on ice for 1 hour and centrifuged at 3000g for 30 minutes at 4 ° C to sediment the proteins that precipitate at 30% of ammonium sulphate. 100% ammonium sulfate was added to the supernatant to obtain a final concentration of 80% and the samples were incubated again on ice for 1 hour and centrifuged as indicated above to sediment the 80% precipitate proteins with sulfate. Ammonium The supernatant containing the 80% precipitated ammonium sulfate proteins was resuspended in 600 µl homogenization buffer. The protein concentration was determined by means of the Coomassie Plus Reagent test kit (Pierce). The homogenized sediment contained 14 µg of µl protein. Therefore, 8400 µg of proteins were collected from 2 grams of agro-infiltrated tobacco leaves. The homogenized sediment was diluted to 1 µg / µl in a buffer containing 50mM Tris HCl pH 8, 10% glycerol and 100 µM PMSF, divided into aliquots and stored at -80 ° C.

Purification of His-6 labeled protein by affinity chromatography with Ni-NTA:

5 grams of agro-infiltrated tobacco leaves were collected and homogenized in 15ml of ice-cold buffer containing Na-phosphate (100mM), Tris HCI pH 8 (10mM), NaCl (200mM), sodium metabisulfite (0.2%), PMSF (1mM), βMercaptoethanol (10Mm). After homogenization, the sample was centrifuged at 6000 g (Beckman rotor, JA 20) for 10 minutes at 4 ° C. Imidazole (10 mM) was added to the supernatant and with NaOH the pH was adjusted to 9. The solution was centrifuged at 42,000 g (Beckman rotor, JA 20) for 60 minutes at 4 ° C. The supernatant was mixed with 1 ml of agarose in Ni-NTA (Quiagen) pre-equilibrated with homogenization buffer + 10mM imidazole pH9 (buffer B). The mixture was homogenized for 2 hours at 4 ° C to allow the protein to bind to agarose beads in Ni-NTA. The beads were compacted on a 1ml polypropylene column (Quiagen) and the resin was washed with 20 ml of buffer B. The protein was eluted with 5 ml (5x1 ml) of buffer B + 250 mM imidazole, pH 9. The aliquots of the fractions were saved to allow enzyme activity during purification. To prevent any inhibition of enzyme activity by the high concentration of imidazole, the eluted fractions were dialyzed against 4 liters of buffer containing Tris-HCl pH 8, 0.1M, 100µM PMSF and 2M ZnCl2 overnight at 4 ºC. Therefore, 1000 g of protein was recovered. The homogenized sediment was diluted to 3gl / enl in a buffer containing 50 mM Tris-HCl, pH 8, 10% glycerol and 100M PMSF, divided into aliquots and stored at -80 ° C. (Dilution D 10,000).

EXAMPLE 2: DEGREATION OF SPECIFIC MONOCATENARY DNA

The activity of recombinant CEL I on single-stranded DNA degradation was performed as described previously (SUNG SC, LAKKOWSKI M Sr. (1962) J Biol Chem. February 1962; 237: 506-11). 30 micrograms of Dnasa, Rnasa and protease without genomic pea DNA were incubated with 2g of protein extract in a buffer containing 50mM Tris-HCl (pH 7.6), 10mM MgCl2, 1mM DTT and 5-PEG-8000 %. To stop the reaction, an equal volume of 20 mM LaCl 3 in 0.2N HCl was added. The samples were centrifuged at 21,000 g for 40 minutes and the absorbance at 260 nm of the supernatant was measured using a spectrophotometer to determine the amount of DNA that had become acid soluble.

EXAMPLE 3: HETERODUPLEX DNA EXCISION AND DETECTION OF A POINT MUTATION KNOWN IN AN AGAROSA TEST GENE AND ACRILAMIDE GELS BY THE ENDONUCLEASE PRODUCED IN TOBACCO

To test whether the CEL I endonuclease produced in tobacco can recognize simple point mutations, the activity of the preparation of recombinant CEL I obtained by precipitation with ammonium sulfate in heteroduplex DNA from two clones differing in a single point mutation was tested: change from CG to AT (Bendhamane et al., Plant Cell, 11, 781-792, 1999). PCR was performed in both clones, using two R21 and R22 oligonucleotides (R21 5 'GAC ATA TGG ACT ACA GAA GCT TGG G 3' SEC ID No. 8; R22 5 'GTT CAC GGG TCA CAT CAT GCA TTC C 3' SEQ ID N ° 9). PCR amplification and reconstitution of heteroduplex DNA was performed using the following program: denaturation for 2 min. at 95 ° C followed by 7 cycles with 20 s at 94C, Tf (55 ° C) +3 ° C at Tf -4 ° C for 5 s, -1 ° C per cycle, gradient at 72 ° C at 0.5 ° C / s and an extension at 72 ° C for 1 min, then 44 cycles with 20 s at 94 ° C, Tf -5 ° C at 30 ° C, gradient at 72 ° C at 0.5 ° C / s, and an extension at 72 ° C for 1 min, a final extension at 72 ° C for 5 min and a denaturation stage at 94 ° C for 10 min followed by an increase of 40 ° C for 20 s and -0.3 ° C per cycle.

The PCR products (a mixture of wild-type and mutant DNA or only wild-type or mutant DNA) were incubated with the preparation of CEL I (stock solution at 1g / dilul diluted to 1/100, 1 / 500 or 1/1000) as follows: for example, 10 µl of PCR product (500 ng) was incubated with 2.5 µl of the reaction buffer (10mm Hepes, 10mM MgSO4, 0.002% Triton X100, KCl 10mm) and 2.5l of the CEL I preparation diluted in a total volume of 25l, for 30 minutes at 37 ° C. The reaction was stopped by 5 µl of 500mM EDTA and the digestion products were analyzed on a 3% agarose gel.

5 As shown in Figure 1, the detection of the point mutation in the test gene, Rx, in heteroduplex DNA was revealed by the appearance of two bands of approximately 200 bp and 400 bp at dilutions 1/100, 1/500 and 1/1000. When enzyme is not added, these bands do not appear.

PCR products were obtained using fluorescently labeled primers and digested in the same manner as before when analyzed on acrylamide gel on an ABI377 sequencer (Figure 2).

As shown in Figure 2, the two 256 and 405 bp bands appear only when the Wt and Mut mutant wild type DNAs have been mixed together due to heteroduplex formation between the two DNAs. This appears even clearer when different wavelengths are used for gel visualization. In particular, the band

15 of 256 bp, labeled with the fluorochrome ROX (red) and not very clearly individualized on the black and white in Figure 2, is clearly present when using the Wt + Mut mixture as a template for PCR and absent in the others cases.

These results demonstrate that CEL I protein produced in plants can recognize mismatches in DNA.

EXAMPLE 4: DETECTION OF PUNCTUAL MUTATIONS IN GENEIC GUIANT DNA BY CEL I RECOMBINANT

25 To test whether purified recombinant CEL I of tobacco could be used to detect single point pea mutations, different rms and pea mutants were used, previously characterized by Catherine Rameau (MORUS et al Plant Physiol 2001, 126: 1205-1213; RAMEAU et al, Plant Physiol 2002, 115: 458-467) as an assay were used; rms1.11 containing G ----> A at different positions in the gene sequence; rms 112 containing G ----> A; rms 1-13 containing G ---> A. They all mutated to G ----> A in the same rms1 gene but in

30 different positions.

To amplify the wild-type and mutant alleles of the rms locus (rms1-13, rms1-10, rms1-12) and the locus le, fifty nanograms of pea genomic DNA were used as a template. The primers used in this PCR amplification are summarized in Table 4.

35

TABLE 4:

Pea mutant name

Primer Name Sequence 5 ’-> 3’

rms 1-10

4m118 5’TTGGTTGGACTTCACTTTGAGC 3 ’SEQ ID N ° 10

4m984

5 ’CACAACAATCAGCAATGACAGC 3’ SEQ ID N ° 11

rms 1-12

4-347 5 ’GTGATTGCTCCACCTCCGCCACC 3’ SEQ ID N ° 12

4-134

5’TACAGCGATTGATATAATATAAAATTATCC3’SEC ID No. 13

rms 1-13

4-960 5 ’GTGTTTGTCCAGTAATAGTGTCAGCATA 3’ SEQ ID N ° 3

4-721

5 ’AGGAACCTGAGAAAAGACTCGCCAGC 3’ SEQ ID No. 4

Ie 1

Ie 2462 5 ’TGATATTGTCGTGCAATATGATGAAAC 3’ SEQ ID N ° 14

Ie 3082

5’ATACCTATTTAGCCCACTTGGACAC 3 ’SEQ ID N ° 15

The PCR amplification program was 94 ° C, 1 min, (94 ° C 15s, 55 ° C 15s, 74 ° C 1min, X35) 74 ° C 7min,

40 8 ° C. The PCR products were analyzed on agarose gel and digested, as described in Example 2 above, with different dilutions of recombinant CEL I preparation obtained by precipitation with ammonium sulfate. As shown in Figure 3, the size of the PCR product in the rms mutant 1-13 and the wild type mutant is approximately 500 bp (exactly 481 bp). As a result of the detection of the heteroduplex DNA mutation, two bands of approximately 200 and 300 bp were obtained. The two bands can

45 be observed on an agarose gel even at a 1/1000 dilution of the protein produced in tobacco. This result demonstrates that the protein produced in plants can first, recognize a point mutation present in genomic DNA and secondly, it is very active since digestion products can be observed even if the protein is diluted to 1/1000.

50 EXAMPLE 5: EFFECTIVENESS OF WRONG MATCH EXCISION IN DIFFERENT WRONG MATCHES BY CEL I RECOMBINANT

To test whether the recombinant CEL I prepared in accordance with the invention preferentially cleaves an erroneous pairing type, such as celery purified CEL I, a series of mutants based on the Rx gene was created. 55 For this purpose, different primers were designed (called Rx-A, T, G or C) each containing a mutation

punctual different. Each of these primers allows one of the 4 bases (A, T, G or C) to be introduced at a given position in the Rx gene. Heteroduplexes were created by mixing the amplification products obtained with the different primers.

The PCR mixture (in total volume of 50 µl) contained the DNA template (50 ng), dNTP (0.2 mM), 5 µl of Pfu PCR buffer (10X, Stratagene), primer Rx 21 (0.4 M), primer Rx-A or Rx-T or Rx-G or Rx-C (0.4M), Pfu (5U, Stratagene, 2.5 Units / l). The program used for PCR amplification was 94 ° C 1min, (94 ° C 15s, 55 ° C 15s, 74 ° C 2min, X35) 74 ° C 7 min, 8 ° C overnight. For the PCR, fluorophore labeled oligonucleotides IRD700 and IRD800 (MWS®) were used to allow detection of erroneous pairings in LICOR4300 (LICOR ®).

The PCR products were analyzed on agarose gel and cloned into pGEM 3Zf. All clones had been sequenced to ensure that the correct mutation had been inserted. To reconstitute all types of mismatch, combinations of these constructs were used as a template in PCR amplification.

The mismatch PCR product incubated as described in Example 2 above, with a recombinant CEL I preparation obtained from tobacco leaves by precipitation with ammonium sulfate as described in Example 1 above.

The products of the digestions were analyzed on acrylamide gel. A 6.5% denaturing acrylamide gel was used and the electrophoresis conditions were: 1500V, 40W, 40mA, 45 ° C with a scan rate of 1.

The results are shown in Figure 4 (Liquor Gel).

These results demonstrate that the CEL I preparations of the invention recognize all types of mismatches and particularly mismatches indicated in the prior art that were weakly recognized by CEL I purified directly from celery. In addition, as shown in Table 2 above, the preparation of CEL I of the invention recognizes mismatches, weakly recognized by the preparation of CEL I of the prior art, such as T / T, G / A, A / G, G / T, T / G with very high specificity, with the preference of erroneous pairings of this enzyme being the following: T / G  A / G  G / G  G / T> T / T  G / A  A / A  C / C> T / C  C / T  A / C  C / A.

This specific activity was also enhanced when the CEL I reaction buffer was supplemented with 5% PEG (data not shown).

EXAMPLE 6: SENSITIVITY OF THE RECOMBINANT CEL I

To verify that the recombinant CEL I of the invention can be used for high-performance genotyping, it was tested to see if it could recognize mutations in a group of individuals.

Genomic DNA with a SNP (single nucleotide polymorphism) (C -> T), corresponding to the dwarf pea mutant Le1, and the genomic DNA of the Torstag culture variety of the wild-type pea was used to amplify the Le1 locus . This genomic DNA had been used as a control for the formation of homoduplex and heteroduplex.

To create heteroduplex DNA, genomic DNA derived from the homozygous pea plant for locus le1 is diluted with homogenous pea plant-derived genomic DNA for locus le1 in different proportions and 30 ng were used for PCR amplification using the T24 fluorochrome-labeled primers (5’TGATATTGTCGTGCAATATGATGAAAC3 ’SEQ ID N ° 14) and the fluorochrome-labeled ROX (MWG®) (5’ATACCTATTTAGCCCACTTGGACAC-3’ Le3082) SEQ ID No. 15). The PCR reactions were performed as follows: 94 ° C 1min, (94 ° C 15s, 55 ° C 15s, 74 ° C 1 min) X35, 74 ° C 7 min. In the test of detection of erroneous pairings, they were used, as a template, DNA heteroduplex reconstituted from PCR products, as written above.

The results of this group experiment using the protein produced in tobacco are presented in Figure 5. As expected, the recombinant protein of the invention can at any time recognize the Le1 PSN of a heteroduplex that has formed between a wild type chain and a mutant DNA chain. As a result of digestion, two bands of expected size were obtained (300 bp and 338 bp) and also a third band corresponding to the undigested homoduplex (638 bp). The 338pb band, which is marked in red, clearly appears on the gel (but is less visible in black and white). More interestingly, the protein can detect the mutation even when a group of 25 different genomic DNAs (addition of Le1 and wild type with 23 unknown genomic DNAs) was used.

Finally, the protein produced in the plant has a high sensitivity, which allows the identification of a known PSN with amplified DNA sequences from genomic DNA of at least 24 individuals.

EXAMPLE 7: BIOINFORMATION RESEARCH FOR DIFFERENT ENDONUCLEASES IN ARABIDOPSIS.

5 An analysis of a family of Arabidopsis genes encoding endonucleases was performed, using the PF02265 protein profile of the PFAM database. This HMM profile was used as a probe to direct all 27117 predicted proteins in the Arabidopsis genome and the entire genome was translated into the 6 different lags of the reading frame to avoid the effect of automatically structural annotation. In this analysis you

10 identified 5 candidate genes At1g11190, At1g68290, At4g21590, At4g21585 and At4g21600.

EXAMPLE 8: CLONING AND EXPRESSION OF GENES CANDIDATES OF ARABIDOPSIS IN TOBACCO LEAVES

15 The cDNA of each candidate gene was amplified by PCR and inserted between the 35S promoter and the VMCa transcriptional terminator in the binary vector pBin61 to create pBIN35S-ENDO1, -ENDO2, -ENDO3, -ENDO4 and ENDO5 corresponding to At1g11190, At1g68290, At4g21585, At4g21590 and At4g21600, respectively (Table 5). These constructs were transformed into Agrobacterium strain C58C1 carrying the virulence auxiliary plasmid pCH32 (HAMILTON CM, et al. (1996) Proc Natl Acad Sci U S A., 93 (18): 9975-9). PCH32 plasmid

20 expresses VirG and VirE and was used to enhance T-DNA transfer. Agrobacterium cells were inoculated in 2 ml of medium with L broth (SAMBROOK et al. 1989) supplemented with 50g / ml of kanamycin and 5g / ml of tetracycline and cultured at 28 ° C overnight. The cells were precipitated and resuspended to a final DO600 concentration of 0.5 in a solution containing 10mm MgCl2, 10mM MES, pH 5.6 and 150 µm of acetosyringone. The cultures were incubated at room temperature for 2 h. before leaf agroinfiltration

25 by Nicotiana benthamiana. Agrofiltered Nicotiana benthamiana plants were incubated for at least 24 hours at 24 ° C, 16 hours of light, 60% humidity. To test the effectiveness of agroinfiltration, the plants were also agro-infiltrated with a construct that expressed the green fluorescent protein (GFP). Before collecting the leaves, the intensity of GFP expression for each sheet was measured using a UV lamp. Only the leaves of the plant expressing the GFP were collected. Candidate proteins were extracted by sulfate precipitation

30 ammonium as described in Example 1.

Table 5: Sequences of the primers used for the cloning of the candidate endonuclease with or without Histidine tag

Candidate proteins

AGI genes Primers

ENDO5

At4g21600 Direct: AAGGATCCGAAAGCTCTGTGTTTCAGA SEC ID N0: 16 Reverse: GGAGTTGTTACGTGGGTTCTCAAGGATC SEQ ID NO: 17

ENDO4

At4g21585 Direct: CTGGATCCCTGTTTTTAACTTTGGAAAG SEQ ID NO: 18 Reverse: GGATGTTCAAGTGATTCTCCTGGATC SEQ ID NO: 19

ENDO3

At4g21590 Direct: AAGGATCCATTCGACAAACTTTGTAAC SEQ ID N0: 20 Reverse: AGAGTGGTCTTGGGAATATTTATCTCAG SEQ ID NO: 21

ENDO2

 At1g68290 Direct: ACGGATCCCATTTCAAAGAACTCTGA SEQ ID NO: 22 Reverse: GACCAATCATTATGCTGTAACTTCAG SEQ ID NO: 23

ENDO1 SEQ ID NO: 2

At1g11190  Direct: CAGGATCCAAGTTTCAAACTTGAAG SEQ ID NO: 24 Reverse: CGGTATGTCGGGTTTGGTTCAAGTGG SEQ ID NO: 25

35 EXAMPLE 9: BIOCHEMICAL CHARACTERIZATION OF CANDIDATE PROTEINS

As a simple test to see if the candidate protein is active, some supercoiled plasmid was incubated with different dilutions of protein extract (80% ammonium sulfate precipitate). As a result of the presence of an endonuclease in the protein extract, the super-rolled structure must relax and some new bands of DNA will appear when the incubation medium is processed on an agarose gel. Using the different solutions you can compare the different endonucleases and see which one is the most active. The control

it was always recombinant CEL I endonuclease prepared by precipitation with ammonium sulfate as described in Example 1 above.

To explore the candidate proteins that can cleave the heteroduplex DNA at the mismatch site, a rapid characterization system based on three consecutive steps was also used.

In the first stage the candidate proteins were tested to determine their ability to degrade single stranded DNA. This condition was tested because the DNA at the mismatch site in the heteroduplex DNA is single stranded. Therefore, an endonuclease that cannot digest single stranded DNA is assumed to be unable to cleave the DNA at the mismatch site. Second, the candidate protein must cleave a test heteroduplex DNA at a well-known and well-characterized mismatch site. Third, the proteins that pass test 1 and 2 were evaluated to determine their effectiveness in cleaving heteroduplex DNA fragments that carry all types of mismatches. This was done using DNA tools that comprise a group of well-characterized plasmid constructs containing in each specific position of the insert, each of the four possible nucleotides.

Ability to degrade a single stranded DNA

The activity of the candidate proteins on the degradation of a single stranded DNA was performed as described in Example 2. In this analysis the five complete candidate proteins showed nuclease activity and were classified from the most active to the least active as follows. : ENDO1, ENDO5, ENDO2, ENDO3 and ENDO4.

Heteroduplex DNA cleavage at C-A / T-G mismatch sites

To test whether candidate proteins produced in tobacco could recognize simple point mutations, the activity of protein extracts in the heteroduplex DNA created as described in Example 3 was tested. The PCR products containing each or both of the alleles were incubated with a 1/1000 dilution of protein extracts derived from agroinfiltrated leaves either with the candidate endonuclease gene or with the GFP as a control, as follows: 500 ng of PCR products were incubated with extract of tobacco protein in a final volume of 25l containing 50mM Tris-HCl (pH 7.6), 10mM MgCl2, 1mM DTT and 5% PEG-8000 for 30 minutes at 37 ° C. The reactions were stopped with EDTA at the final concentration of 80mM and analyzed on a 3% agarose gel.

If the two oligonucleotides were fluorescently labeled, the digestion products were analyzed on acrylamide gel, in a 377Abi DNA sequencer. In this experiment, it was predicted that if the protein extract contained a specific mismatch endonuclease, the heteroduplex DNA could cleave the mismatch site, thereby releasing two bands of 256 bp and 405 bp.

The results are shown in Figure 6 (note that the 256 bp band, marked in red, is clearly visible when the enzyme is in the presence of heteroduplex and otherwise absent; this band differs less in black and white).

From this biochemical analysis, it is concluded that the five endonucleases show specific cleavage activities of mismatch. In addition, the three enzymes, ENDO 1, ENDO 5, ENDO 2, which have higher nuclease specific activity for single stranded DNA, also more effectively cleave heteroduplex DNA at the C-A / T-G mismatch site. In addition, among these three endonucleases, ENDO 1 and ENDO 5 were the most active. Therefore, these two nucleases were selected for a more precise characterization.

Efficacy of cleavage of mismatch by candidate endonuclease in different mismatches

The main objective of this work is to identify an endonuclease that cleaves mismatches that CEL-I does not recognize effectively. This was done using DNA tools that comprise a group of well-characterized plasmid constructs containing each of the four possible nucleotides at the specific position of the insert. To reconstruct all types of mismatch, the combination of these constructs in PCR amplification was used as a template.

The mismatch PCR product was incubated as indicated above (see example 5) with the candidate endonuclease and analyzed on the LICOR sequencer.

The results are shown in Figure 7.

In this analysis, CEL 1 and ENDO 5 weakly recognize the type of mismatch T / T. On the other hand, ENDO 1 recognizes almost all types of mismatch with high efficiency. From this analysis, it can be concluded that ENDO 1 can recognize mismatches not detected by CEL-1.

EXAMPLE 10: SENSITIVITY OF ENDO 1 TO DETECT A MUTANT ALELUS IN A DNA GROUP.

The sensitivity of ENDO 1 was evaluated as described in Example 3, with dilutions of 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 50 or 60 times with DNA carrying the allele of wild type.

In Figure 8, the results of this collective experiment are presented, which shows the detection of an allele from a homozygous mutant among 60 alleles from a homozygous wild type. The PCR of the two organisms was performed and the amplicons were quantified on agarose gel and mixed together following a decreasing ratio of 1: 1 to 1:60 of the PCR amplicons. Then, the activity of ENDO 1 was tested in these different proportions of mutants: wild type.

In identical experiments, CEL I purified from celery can detect an allele in a maximum of 16, with low sensitivity and only a correct sensitivity is obtained when the dilution is 8 times lower or equal.

In summary, the endonucleases according to the invention and particularly ENDO 1 have a much lower reference interference than CEL I, can be used at very high dilutions compared to CEl I and have better specificity and activity than the CEL I endonuclease.

Point mutation detection in acrylamide gel.

A series of mutants based on the Rx gene was created. Different plasmids were designed each containing a type of mismatch to demonstrate the specificity of ENDO 1. The PCR mixture contained specific oligonucleotides for plasmids. For the PCR, oligonucleotides labeled with ROX and FAM fluorophores (MWG) were used to allow detection of mismatches in ABI377 MVG.

As shown in Figure 9, ENDO does not cleave when there is only homoduplex, for example line 1 or line 5 and the only bands that can be seen on the gel are homoduplex on the top of the gel (approximately 600 bp).

At any time an erroneous pairing is obtained in the samples of this document (resulting from the formation of heteroduplex between two strands of DNA from 2 different plasmids, as for example in lines 2, 3, 4 or 6, 7 and 9) the ENDO 1 recognizes and cleaves at the site of erroneous pairing, resulting in the appearance of two bands corresponding to the two products, each marked with a fluorophore. For digestion with ENDO 1, a decrease in the reference can be observed and only some mismatches with ENDO 1 can be detected (line 9 for example).

Detection of a known point mutation in ENDO 1 acrylamide gel in two different dilutions D 1000 and D 5000.

Two plasmids containing a wild form and a mutated form of the Rx gene were used. These differ only in a known point mutation. The PCR mixture contains specific oligonucleotides for plasmids. Oligonucleotides labeled with the ROX and FAM fluorophores (MWG) were used in the PCR to allow detection of erroneous pairing in ABI377 MWG.

As shown in Figure 10, at any time there is a heteroduplex in the samples of this document (M5 + M12), ENDO 1 can recognize and cleave at the wrong pairing site, resulting in the appearance of two bands marked with a fluorophore When only homoduplex M5 or M12 are present, ENDO 1 does not cleave and the only bands that can be seen in the gel are homoduplex in the upper part of the gel (approximately 600 bp). When D 5000 is used for ENDO 1, an improvement in the reference can be observed. Therefore, ENDO 1 can use a very high dilution compared to any of the nucleases known in the art.

SEQUENCE LIST

<110> GENOPLANTE-VALUE NATIONAL INSTITUTE OF AGRICULTURAL RESEARCH BENDAHMANE, Abdelhafid STURBOIS, Bénédicte TRICKS, Karine CAPE, Michel

<120> METHOD FOR PRODUCING HIGHLY SENSITIVE ENDONUCLEASES, NEW ENDONUCLEASE PREPARATIONS AND USES OF THE SAME.

<130> MJP / bv1516-19

5

<150> PCT / EP2004 / 009159 <151> 07-30-2004 <150> PCT / EP2004 / 009166 <151> 07-30-2004

10

<160> 25 <170> PatentIn version 3.3

fifteen

<210> 1 <211> 2640 <212> DNA <213> Arabidopsis thaliana

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<220> <221> misc_feature <223> Gen At1g11190 of ENDO 1

<400> 1

image 1

image 1

<210> 2

<211> 305 5 <212> PRT

<213> Arabidopsis thaliana

<220>

<221> misc_feature 10 <223> At1g11190 of the ENDO 1 protein

<400> 2

image 1

image 1

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 4-960

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<210> 4

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 4-721

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<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> CEL N-terminal

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<210> 6

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> C-CEL terminal

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<210> 7

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> C-terminal with His 8 amino acid CEL <400> 7

image 1

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer (R21)

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<210> 9

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer (R22)

image7

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> 4m118 primer

image8

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 4m984

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<210> 12

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 4-347

image10

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 4-134

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<210> 14

<211> 27

<212> DNA

<213> Artificial sequence

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<223> Primer Ie 2462

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<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer Ie 3082

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<210> 16

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Direct primer for ENDO5

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<210> 17

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for ENDO5

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<210> 18

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Direct primer for ENDO4

<400> 18 <210> 19

image 1

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for ENDO4

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<210> 20

<211> 27

<212> DNA

<213> Artificial sequence

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<223> Direct primer for ENDO3

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<210> 21

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for ENDO3

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<210> 22

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Direct primer for ENDO2

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<210> 23

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for ENDO2

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<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Direct primer for ENDO1

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<210> 25

<211> 26

<212> DNA

<213> Artificial sequence 10

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<223> Reverse primer for ENDO1

image22

Claims (15)

1. A method for producing a specific recombinant endonuclease of mismatches in which said method comprises: - expressing said recombinant endonuclease in cells of a complete host plant or of a separate organ thereof, transiently transformed by agroinfiltration with an Agrobacterium strain containing an expression vector comprising a polynucleotide encoding said endonuclease; - isolating said recombinant endonuclease from said cells of the host plant.

2.

A method according to claim 1, wherein said agroinfiltration is performed on a sheet of said host plant.

3.

 A method according to any of claims 1 or 2, wherein said host plant belongs to the genus Nicotiana.

Four.

 A method of any one of claims 1 to 3, wherein said endonuclease is isolated from the organ of the agro-infiltrated plant by a process comprising the following steps:

- extracting the content of the cells from the agroinfiltered organ that expresses said endonuclease; - add to said extract ammonium sulfate at a final concentration of at least 30% and separate the protein precipitate from the supernatant; - adding to said supernatant ammonium sulfate at a final concentration of at least 80% and recovering the protein precipitate containing the endonuclease.

5.

The method according to claim 4, wherein the ammonium sulfate is added to a final concentration of 30% in the first stage of precipitation and a final concentration of 80% in the second stage of precipitation.

6.

A recombinant endonuclease preparation specifies of mismatches that can be obtained by the method of any one of claims 1 to 5, wherein said endonuclease is selected from CEL I of Apium gravoeolens and BFN1 of Arabidopsis thaliana of SEQ ID NO: 2.

7.

A recombinant CEL I endonuclease preparation of claim 6, wherein said recombinant CEL I endonuclease has the following preference for mismatches: T / T ∼ T / G ∼ A / G ∼ G / G ∼ G / A ∼ G / T ≥ A / A ∼ C / C ≥T / C ∼ C / T> A / C ∼ C / A and can recognize a mutant allele in the presence of 23 times the wild type allele.

8.

 A recombinant BFN 1 endonuclease preparation of claim 6, wherein said recombinant BFN 1 endonuclease has the following preference for mismatches: G / G ∼ G / A ∼ A / G ∼ G / T ∼ T / G> T / T ∼ A / A ∼ C / C ∼ T / C> C / T ∼ A / C ∼ C / A, and can recognize a mutant allele in the presence of 59 times the wild type allele.

9.

The use of a recombinant endonuclease preparation according to any one of claims 6 to 8, to detect in a DNA duplex, a mismatch resulting from a base replacement or insertion or deletion of one or more nucleotides in a chain of said duplex.

10.

The use of a recombinant endonuclease preparation according to any one of claims 6 to 8, in an erroneous pairing cleavage protocol by detection of locally induced genome lesions (TILLING).

eleven.

The use of a recombinant endonuclease preparation according to any of claims 6 to 8, for the identification of DNA polymorphisms in natural populations, by Ecotilling.

12.

The use of a recombinant endonuclease preparation according to any one of claims 6 to 8, as a mismatch detection reagent.

13.

The use of a recombinant endonuclease preparation according to any one of claims 6 to 8, in a method of selecting mismatches.

14.

The use of a recombinant endonuclease preparation according to any one of claims 6 to 8, to simultaneously select one or more mutations in a target gene in a population of any organism or a cell line derived therefrom performing the steps of :

a) amplifying said target gene or part thereof for each individual of said population, b) classifying said amplification products in a bi or three-dimensional matrix, comprising lines, rows (two-dimensional matrix) and columns (three-dimensional matrix), c) regrouping said amplification products, to obtain different groups, each group representing a row, a line or a column of said matrix, d) adding to each group a reference amplification product obtained from a non-mutated gene and incubating said groups under conditions that allow the formation of heteroduplex, ee) incubate each group with said endonuclease preparation, and f) detect the presence of heteroduplex in said incubated groups.

fifteen.

The use of a recombinant endonuclease preparation according to any one of claims 6 to 8, to simultaneously select one or more mutations in a target gene in a population of any organism or cell line derived therefrom, performing the steps of :

a) classify each individual of said population in a two- or three-dimensional matrix comprising lines, rows (two-dimensional matrix) and columns (three-dimensional matrix), b) group each row, line and column to obtain different groups, therefore representing each group a row, line or column of said matrix, c) add to each group a reference gene product obtained from a non-mutated gene, d) amplify said target gene or part thereof in each group to achieve product groups amplified, e) incubating said amplified product groups under conditions that allow the formation of heteroduplex, f) incubating said amplified product groups with said endonuclease preparation, and g) detect the presence of heteroduplex in said incubated groups.