WO2013038101A1 - Method for analysing genomic dna - Google Patents

Abstract

The present invention relates to a nucleic acid sequence with sequence SEQ ID NO: 1, and to the use of same for analysing human genomic DNA, in particular as a universal internal calibrator. It also relates to a method for in vitro analysis of the variation in the genomic quantity of a target gene, and to the use of said method for various applications: for assessing the quality of a sample of human DNA, carrying out a molecular analysis of a tumour, determining the effect of a substance or a physical and/or biological treatment on the number of copies of a target gene in a sample, finding, in a substance screening test, those which positively or negatively regulate said number of copies of target genes, or carrying out a tumour screening test. The present application further relates to a human genomic DNA analysis kit comprising, in particular, sense and antisense primers for amplifying the sequence SEQ ID NO: 1 and a fluorescent probe for quantifying the SEQ ID NO: 1 amplicons produced.

Description

 METHOD OF ANALYZING GENOMIC DNA

Technical area

 The present invention relates to a nucleic acid sequence, as well as to its use for human genomic DNA analysis, especially as a universal internal calibrator.

 It also relates to an in vitro method for analyzing the variation of the genomic quantity of a target gene, and the use of this method for various applications: to evaluate the quality of a human DNA, to perform a molecular analysis of tumor, determine the effect of a substance or physical and / or biological treatment on the number of copies of a target gene in a sample, search in a screening test of substances, those that regulate positively or negatively this number of copies of target genes, or perform a tumor screening test.

 The present invention also relates to a human genomic DNA analysis kit.

 It finds applications in the field of biotechnologies, for example for the quality control of DNAs distributed by biobanks, for the molecular analysis of tumors, for testing the toxicity and genotoxicity of cosmetological or pharmacological molecules, or for screening in search of drugs.

 In the description below, references in brackets ([]) refer to the list of references at the end of the examples.

State of the art

The variability of the number of copies of a gene is a particular form of polymorphism. It corresponds to the fact that the number of copies of the same gene in the genome is variable between individuals of the same species. It is estimated that more than 12% of the human genome is affected by this type of polymorphism (Redon et al., Global variation in the human genome ", Nature, vol. 444, 2006, p. 444-454 [1]).

 The number of copies of a target gene may also vary pathologically in the case of certain tumors and may give them, then, a particular "marking". Being able to measure this number, can also serve as a tool for screening molecules, to enable one to search for the beneficial or harmful activity of a substance. It also makes it possible to screen tumors for correlations between the number of gene copies and therapeutic treatments. Finally, it also makes it possible to measure directly in a given DNA the natural or artificial degradation of this DNA.

 The variation in the number of copies of a target gene can be a powerful genetic marker for the diagnosis of a large number of genetic diseases, such as trisomy, or cancers or even resistance to cancers (breast cancer and amplification). of CerB2).

 The variation in the number of copies of a target gene can also make it possible to measure the variation in the number of mitochondria and therefore the variation in the metabolic activity of a cell. It can thus be a toxicological indicator.

 It can also be an indicator of the chances of responses to a therapeutic treatment or an indicator of the evolution of a disease, such as cancer, during a therapeutic treatment.

The European Union's REACH ("Registration, Evaluation and Authorization of Chemicals") Ordinance, which entered into force in June 2007, obliges the chemical, cosmetic and pharmacological industries to provide health and environmental safety data on all the substances they produce. The provision of these data may include the use of DNA analysis methods. The methods currently used to quantify a DNA are optical density measurement and agarose gel electrophoresis. These methods are crude and do not allow to appreciate the quality of the DNA and in particular its minimal degradation. More recently, trials have been proposed to quantify double-stranded DNA by intercalating Syber green (registered trademark). However, the results obtained are very unstable. Another method used is UV spectrometry. However, this method also poses problems of interpretation of the results because of the significant difference in absorption between single-stranded and double-stranded DNAs.

 Moreover, to analyze genomic DNA, the methods currently used are fish ("fluorescence in situ hybridization"), allelotyping, CGH array ("comparative genomic hybridization chip") and possibly massive sequencing. . These methods require the use of expensive equipment and hyperspecialized personnel for the interpretation of the results. In addition, the results obtained by these methods are not quantitative.

 There are therefore real needs to develop new tools for genomic DNA analysis, including new methods, overcoming the defects, disadvantages and obstacles mentioned above in the prior art. In particular, it is necessary to develop a process that is easy to implement, inexpensive, adapted to broadband and has good sensitivity and good specificity. Description of the invention

The present invention responds precisely to these needs by providing a method which makes it possible to measure the intact genomic quantity, for example the number of copies, of any target gene compared to a universal internal calibrator, as well as the means necessary for the implementation. implementation of this process. Thus, the present invention particularly relates to a nucleic acid of SEQ ID NO: 1 sequence.

 It also relates to the use of the nucleic acid SEQ ID NO: 1 for a human genomic DNA analysis.

 According to the invention, the nucleic acid SEQ ID NO: 1 can be used, for example, as a calibrator for a human genomic DNA analysis.

 As used herein, the term "calibrator" means any nucleic acid sequence for normalizing the results of a genomic DNA assay. The term "calibrator" can also be used.

 The inventors of the present are the first to have identified the sequence SEQ ID NO: 1 as being a nucleic acid highly conserved in humans, that is to say extremely stable, that is to say without variation number of copies, in the human genome. This sequence is located in the RXRB region in the first intron of Chromosome 6. This sequence has been designated by the inventors as "universal internal calibrator".

 By nucleic acid sequence SEQ ID NO: 1 is meant a sequence consisting of the sequence SEQ ID NO: 1 or a sequence having the same function and having at least 60% homology with SEQ ID NO: 1, for example at least 70%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99%.

 Moreover, the inventors have found that the number of copies of this sequence in the human genome is constant, namely approximately

330 haploid copies / ng of DNA.

In the present invention, the nucleic acid of sequence SEQ ID NO: 1 may also be called "RXRB gene", "RXR beta gene", "betaanan", "calibrator according to the invention", "internal calibrator, or" universal internal calibrator ". In the present, the term "human genomic DNA", the assembly comprising the human chromosome genome and the human mitochondrial genome.

 The term "human chromosomal genome" means all the genetic material contained in the chromosomes of a human being.

 The term "human mitochondrial genome" means all the genetic material contained in the mitochondria of a human being.

The subject of the present invention is also an in vitro method for analyzing human genomic DNA using the nucleic acid of sequence SEQ ID NO: 1.

 The method according to the invention may especially be an in vitro method for analyzing the variation of the amount of a target gene different from SEQ ID NO: 1 comprising the use of a nucleic acid of sequence SEQ ID NO: 1 .

 For example, the in vitro method for analyzing the variation of the quantity of a target gene different from SEQ ID NO: 1 comprises a step of comparing the quantity of the target gene in a test sample with the quantity of the target gene. in a reference sample.

 For example, the step of comparing the amount of the target gene in a test sample with the amount of the target gene in a reference sample can be performed using the ΔΔ method of Ct.

 The ΔΔ method of Ct is a method well known to those skilled in the art (CA Heid, Stevens J, KJ Livak, and Williams PM Real time quantitative PCR, Genome Res 1996. 6: 986-994 [2]; Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, MW Pfaffl, Shipley GL, Vandesompele J, Wittwer CT The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments Clin Chem 2009, 55 (4): 61 1 -22 [3] Weglarz L, Molin I, Orchel

A, Parfiniewicz B, Dzierzewicz Z. Quantitative analysis of the level of p53 and p21 (WAF1) mRNA in human colon cancer HT-29 cells treated with inositol hexaphosphate. Acta Biochim Pol. 2006; 53 (2): 349-56 [4]. It uses the following mathematical relationship:

The AACt method is well known to those skilled in the art for measuring the amount of a cDNA in a given sample relative to a reference cDNA. We have adapted this technology to measure the number of genomic copies of a given target gene compared to the number of genomic copies of a calibrator gene that has the particularity of being very stable. The developed formula is the following ACt (sample to be tested) =

Ct (target gene in test sample) - Ct (calibrator gene in test sample) and ACt (control DNA sample) = Ct (target gene in control DNA sample) - Ct (calibration gene in control sample). The principle of qPCR, its operating protocol, the various settings of the thermal cycler are presented, for example, in the document "Relative Quantification: Data Management & Analysis Settings", Ray Meng, [5] (available online at http://genome. hku.hk/portal/files/GRC/Events/Seminars/2009/20090512/rel ative% 20quantification_data% 20management% 20% 20analysis% 20setting s. pdf).

 The main and particular concepts applicable to the method of analyzing the variation in the number of genomic copies of any target gene in a sample are as follows:

 Ct ("Cycle treshold") is the point, the cycle, of the amplification curve of the reference sample where the detected fluorescence becomes greater than the background noise.

The Ct is determined automatically by the qPCR device used but it must, from time to time, be adjusted manually on certain thermal cyclers. The threshold used ("threshold" in English) is set so as to cut all the fluorescence curves at the exponential phase of the qPCR. In this case, the value of Ct represents the initial amount of DNA template present in the sample.

 The mean reference Ct is calculated using a reference DNA dilution range and will then be used to homogenize measurements possibly made on other PCR plates. Indeed and for each new qPCR plate, the points 100, 50 and 25 ng of a reference DNA range of the laboratory are always analyzed in triplicate for a given target gene. These points are used to ensure the effectiveness of the duplex qPCR concerned and allow the standardization and therefore the comparison of the results obtained handling manipulation for thousands of samples. The average reference Ct obtained with an initial range of adequate DNA dilution will in fact always be used for all subsequent manipulations for the target gene concerned.

 The reference DNAs ideally contain normal target gene and calibrator gene amounts of approximately 330 copies of haploid genome / ng each.

 The amount of target gene for the range of laboratory reference samples is "fed back" into the thermal cycler software as containing 25, 50 ng and 100 ng of target gene. The calibrator has been indicated as "calibrator". Samples are indicated as "standard" or control. These parameters have been chosen in order to be able to use the thermal cycler software that is not made for this purpose.

 For example, the CFX manager software can be used using the following mathematical relationship:

AACt = ACt (sample to be tested) - ACt (standard sample / control) In this formula, ACt (sample to be tested) = Ct (target gene in test sample) - Ct (calibrator gene in test sample) and ACt (standard DNA sample / control) = Ct (target gene in test sample Standard DNA / control) - Ct (calibrator gene in Standard / Control sample).

 The obtained values are transformed into logarithms of the DNA concentration to compare and represent them graphically as in Figure 1 to create a standard range that reads the average Ct calculated by the machine, the slope of the curve and therefore the efficiency of the qPCR duplex gene target / universal calibrator. Samples to be analyzed can be added and analyzed at the same time and on the same plate and the results obtained will be calculated in the same way by including if necessary a control DNA positive or negative of the experiment that generated the DNAs.

The results obtained will be automatically transformed into 2 ^"

^AACt for comparison and finally expressed in automated histograms.

 The histograms indicate on the abscissa: the name of the sample and on the ordinate the number of times [increased (amplification) or decreased (deletion)] of copies of the target gene compared to the number of copies of the target gene measured in the control DNA specific experience. The results are normalized by the calibrator.

Usually, quantization software from qPCR machines is suitable for measuring cDNA concentrations using the AACt method and converting them to 2 ^_ΔΔε . The results are expressed relative to a reference gene (cDNA) whose expression does not vary. The reference gene is measured in each sample relative to a control sample whose amount of cDNA is indicated in the software. A cascading dilution of this control is used to ascertain the efficacy parameters of qPCR. The results are cDNA concentrations.

To use this software in the state for measurement of genomic DNA, the reference gene used is the calibrator according to the invention. The sample control, whose concentration is known, is the standard DNA / control. For example, the fluorescence of the probe of the reference gene, that is to say the calibrator gene is orange. The calibrator makes it possible to normalize all the results obtained for each target gene. For example, the fluorescence of the second probe, which hybridizes to the target gene, is yellow. These fluorescences will be measured at the end of each cycle by the thermal cycler (real time measurement)

Using the AACt method described above and conversion to 2 ^_ΔΔε , the "fluorescence ratio" (e.g., yellow on orange), duplex qPCR, i.e. the target gene on the internal calibrator , is most often equal to 1 in the reference DNA and in all its dilutions because it is the ratio of the yellow to orange fluorescence intensity which is encrypted. It is also in control DNAs. This with a tolerance of plus or minus 5%; all samples with a calibrator greater than 28 are removed and retested.

 The ΔΔ method of Ct is also presented in Example 1 below.

According to the invention, the amount of gene is advantageously a gene copy number. By "number of copies of a gene" is meant the relative or absolute amount of a gene in the genome.

 As used herein, "sample" means any solution comprising DNA. It may be for example a solution comprising a DNA previously extracted from an individual.

 The term "reference sample" means any sample comprising DNA previously taken from a reference individual.

 The term "sample to be tested" or "sample to be analyzed" means any sample comprising DNA previously taken from an individual to be tested.

"Individual" means one or more cells, a tissue, an organ or a living being. For example, the individual can be any cell comprising DNA. For example, the individual may be a HepG2 cell, a normal liver cell, a normal heart cell, a tumor cell, a fetal cell, a stem cell, a genetically engineered cell. It may also be an in vitro reconstituted tissue, for example, it may be a skin explant or an ocular explant, an organ or an organ fragment.

 Reference person refers to any individual who is chosen as a reference or control. It may be for example any individual whose copy number of a given target gene is known. Indeed, a reference individual for a target gene A is not necessarily a reference individual for a target gene B.

 The term "individual to be tested" is understood to mean any individual whose number of copies of a given target gene is desired to be known.

 As used herein, the term "target gene" is intended to mean any DNA sequence included in a genome. A target gene can be determined by its sequence or by sense and antisense primer sequences which make it possible to amplify the sequence of the target gene.

 According to the invention, in the in vitro method for analyzing the variation of the amount of a target gene different from SEQ ID NO: 1, the reference sample and the sample to be tested may come from a single individual. or two individuals, or even more than two individuals. Thus, according to the present invention, the reference individual and the individual to be tested may be the same individual.

 When the reference sample and the sample to be tested come from two individuals, the implementation of the method according to the invention makes it possible to analyze at a given moment the variation over time of the number of intact copies of a gene. target different from SEQ ID NO: 1.

When the reference sample and the test sample come from a single individual, the reference sample and the test sample may, for example, have previously been taken from a single sample. individual and be analyzed at two or more different times. The reference sample and the sample to be tested may also have undergone different treatments. In this case, the implementation of the method according to the invention makes it possible to analyze the variation over time of the number of copies of a target gene different from SEQ ID NO: 1 in the same individual.

 The method according to the invention allows a true quantification of the number of intact genomic copies of target genes using existing machines and software.

 The process according to the present invention may for example be carried out by qPCR (for "quantitative polymerase chain reaction" in English), also called RT-PCR (for "real time polymerase chain reaction" in English), real-time PCR or quantitative PCR. .

 QPCR is a technique well known to those skilled in the art. It requires the use of sense and antisense primers for amplifying a sequence, as well as the use of a fluorescent probe making it possible to quantify the amplicons produced during the qPCR.

 The sense and antisense primers and the fluorescent probe are nucleic acid sequences which are advantageously specific for the sequence to be amplified.

 Moreover, the fluorescent probe may for example have a fluorochrome. Advantageously, this fluorochrome is stable, resistant to pH and temperature variations and does not modify the half-denaturation temperature ("Tm") of the probe. For example, the fluorochrome can be chosen for its energy absorption / emission properties, in accordance with the detection capabilities of qPCR machines.

 For example, the fluorochrome may be selected from the group consisting of cyanines, rhodamines, coumarins. For example, the fluorochrome may be selected from the group consisting of Texas red (registered trademark), Cascade Blue (registered trademark), Alexa Fluor

(registered trademark), VIC (registered trademark), FAM (registered trademark), Yakima yellow (registered trademark), TET (registered trademark), JOE (registered trademark), HEX (registered trademark), ATTO 550 (registered trademark), Tamra (Tetramethylrhodamin) and ROX (registered trademark) .

 A fluorochrome, also called fluorophore, is a molecule that emits fluorescence. It comes from a functional group that in this molecule absorbs the electromagnetic energy of a given wavelength and re-emits it at another specific wavelength. The skilled person is able to determine what wavelength to apply depending on the fluorochrome used. The fluorescence emitted is measured online at the end of each qPCR cycle.

 The fluorescent probe may advantageously have both a fluorochrome and a fluorescence inhibitor, also called extinguisher or "quencher", one being arranged in 5 'of the sequence of the fluorescent probe, the other being arranged in 3' of this same sequence. An extinguisher is a molecular entity, or chemical species, capable of deactivating an excited state created in a molecular entity by energy, electron or chemical transfer. Thus a quencher is a molecule that can inhibit the fluorescence of a fluorochrome.

 For example, the quencher may be selected from the group consisting of BHQ1 ("Black Hole Quencher 1"), BHQ2 ("Black Hole Quencher 2"), Dabsyl ( dimethylaminoazosulfonic acid), Dark Quencher Eclipse (trademark) with BBQ650 (registered trademark), TAMRA (Tetramethylrhodamin), Qxl quenchers (registered trademark), iow black FQ lowa (registered trademark) and black RQ IRDye QC- 1 (registered trademark).

 For example, the fluorescent probe may be a TaqMan (trademark) probe.

Before qPCR, when no amplification has taken place and the fluorescent probe is intact, the quencher inhibits fluorochrome fluorescence. Only residual fluorescence can be observed. At During qPCR, fluorescent probes hybridized on the synthesized amplicons are hydrolysed during each stage of elongation of the primers. The fluorochrome is then released far from the extinguisher and its fluorescence is no longer inhibited by it.

 The emission of the fluorescence released can then be measured at the end of each qPCR cycle. This measurement makes it possible to define a value of "Ct" ("cycle threshold" in English or "threshold cycle") corresponding to the number of qPCR cycles necessary so that the value of the fluorescence intensity is then significantly different from the background noise. that is to say different from the initial initial fluorescence.

 The method of the present invention can be performed at any temperature to analyze genomic DNA. When the target gene to be analyzed is the mitochondrial genome, or even a transfected gene, it is possible to improve the method of the invention, for example by using reaction temperatures which are more unfavorable for these target genes because they are in excess by report to the calibrator and saturate the detection capabilities of the qPCR machine. For example, a primer pairing temperature of between 58 and 64 ° C, for example 63 ° C, may be used.

The qPCR can be carried out in simplex, in different tubes / wells, that is to say that a single target is amplified by reaction of qPCR, or in duplex, that is to say that two different targets are amplified in a single qPCR reaction. It can also be realized in triplex or quadruplex, that is to say that respectively two or three targets are amplified in a single reaction of qPCR in addition to the universal calibrator. Preferably, the primers and probes used are designed and validated to give, together, specific qPCRs and comparable efficiencies.

Preferably, the qPCR is carried out in duplex. In fact, the realization of duplex qPCR makes it possible in particular to save reagents since both qPCRs occur simultaneously in the same tube. This also eliminates variations due to handling errors since both qPCRs occur simultaneously in the same tube.

 When the qPCR is performed in duplex, two fluorescent probes labeled respectively with two different fluorochromes must be used to differentiate the amplicons produced during the qPCR. According to the present invention, when the qPCR is carried out in duplex, it always includes the same universal calibrator, namely the nucleic acid sequence SEQ ID NO: 1.

 When two fluorescent probes are respectively labeled with two different fluorochromes, it can be any fluorophore polyplex which gives detections well separated from the fluorescences. It may be, for example, pairs of fluorochromes selected from the group consisting of Texas red (registered trademark) A / IC (registered trademark), Texas red (registered trademark) / Yakima yellow (registered trademark), Texas red (registered trademark) / TET yellow (registered trademark), Texas red (registered trademark) / HEX (registered trademark), Texas red (registered trademark) / JOE (registered trademark).

 Preferably, two fluorochromes having close quantum yields are used, for example the pair of fluorophores: Texas red (registered trademark) / Yakima yellow (registered trademark).

Several industrial applications are possible, in particular depending on the nature and the games of the target gene or genes chosen, the universal internal calibrator remaining the same, namely the nucleic acid sequence SEQ ID NO: 1.

For example, sets of target genes have been identified by the inventors because they present, alone or together, an interest in diagnosis and / or prognosis. Depending on the target genes analyzed, it is therefore possible, for example: to evaluate the degradation of a DNA. This application is referred to as "DNAqual" herein. It allows for example to test the quality of DNAs such as DNAs extracted from individuals, distributed by biobanks or to quantify the degradation of a DNA caused by physical, biological or chemical phenomena, direct or indirect. Thus, the term "evaluation of the quality of a DNA", the evaluation of the entirety of a DNA, its possible level of degradation. DNAqual also tracks the amount of damage caused by a treatment or a substance or a cell deficit in enzymes that can repair genomic DNA. to measure the number of copies of mitochondrial genome and thus the number of mitochondria present in a cell or a tissue at a given time or at different times. This application is referred to as "MITOC" herein. It also shows the toxicity of a given molecule or treatment by showing changes in the number of mitochondrial genome copies over time. to show the genotoxicity induced by a given molecule or a given treatment when the target genes used border fragile sites of the DNA, and others such as for example sites selected from the group comprising P16, AGAP2, GRID2, MMRN1, LRP1 B, SPOP, FHIT, FNLB, CNTNAP4 and ADAMTS18, for example in the group comprising P16, GRID2, LRP1 B, FHIT and ADAMTS18. A target gene selected from the group consisting of human EGFR1, human TNFRSF10A (trail receptor 4), human TNFRSF10B (TRAIL-R2 - CDC262), human TPBG, human ERBB3, and human ERBB4 can be used as a negative control. This application is referred to as "Genotox" herein. For example, the Genotox application can be performed on extracts of cells or tissues in the purpose of assessing their levels of intoxication. Genotox can also be produced from cells or model tissues treated by one or more potentially toxic physical, chemical or biological phenomena (such as a protein) in order to appreciate the possible mutagenic effect of the phenomenon tested on the genome of the cells. . Indeed, a mutagenic effect is manifested by an unfaithful replication of the DNA damaged by the toxic product and therefore variations in the number of copies of these genes, whether deletions or amplifications. In theory, they are more likely to strike the DNA of fragile sites. For example, Genotox can be used in a screening test for substances that could have a toxic effect on an individual. For example Genotox can be used in a screening test of cells or tissues that have been subjected to different treatments to evaluate the toxicity of these treatments. In another example, Genotox can be used in a test on cells or tissues that have been subjected to different treatments to classify these cells or tissues according to their degree of resistance to these treatments. to allow a molecular categorization of pathologies, for example tumors, in the context of personalized medicine, for example, to better indicate a therapeutic by measuring the number of copies of target genes for which there is a therapeutic targeting these genes, their proteins or corresponding cellular pathways. For example, there are therapies targeting one or more molecules, for example receptors involved in the disease, by means of monoclonal antibodies or small chemical molecules. To predict, follow or understand the resistance of tumors to therapies such as targeted therapies or chemotherapies because these tumors have amplifications of the genes involved in such resistance (eg MDR, Topo 1 and 2) by measuring the number copies of these resistance genes. To predict, follow or understand the aggressive nature of a pathology, for example a tumor, and the chances of responding to a treatment by detecting the aggressiveness signatures of this pathology, for example a tumor. This application is referred to as "Copycount" herein.

Copycount could therefore have utility as part of diagnostic kit and / or theranostics, to help identify the best treatment and monitor the disease during treatment (companion treatment test). By "theranostics" is meant the combination of a diagnostic test with a therapy. For example,

Copycount could be used in a tumor screening test. Tumor screening with Copycount, for example, makes it possible to evaluate the link between the effectiveness of a therapeutic treatment and the copy numbers of the genes coding for the biological target of the therapeutic treatment.

to quantify the results of transfection experiments of a luciferase plasmid in model cells or tissues. This application is referred to as "Luciola" in this.

- to measure signatures of tumor aggressiveness and ethnicity.

In addition, the inventors have developed sense and antisense primers, as well as fluorescent probes specific for each of these target genes. These target genes, sense and antisense primers and fluorescent probes are shown in Table 1 below. The sequences of the sense and antisense primers developed by the inventors for amplifying the calibrator of sequence SEQ ID NO: 1 are respectively the sequences SEQ ID NO: 2 and SEQ ID NO: 3. The specific fluorescent probe developed by the inventors to detect amplicons products of the nucleic acid sequence SEQ ID NO: 1, has the sequence SEQ ID NO: 4.

Table 1: Possible applications in function of the target genes

 analyzes

The subject of the present invention is also the use of the in vitro method for analyzing the variation of the quantity of a target gene different from SEQ ID NO: 1 defined above for evaluating the quality of a human DNA.

 In the present context, the term "evaluate the quality of a DNA" is intended to quantify the degradation of a DNA generated by physical and / or biological and / or chemical phenomena. For example, the physical phenomena can be a UV irradiation of the DNAs, a prior lyophilization of the tissues, a heating of the DNAs, etc. The biological phenomena can be for example a DNA degradation caused by a microorganism, for example a bacterium or by an enzyme. The chemical phenomena can be for example any phenomenon caused by a substance, a molecule in a cell / model tissues, etc. It can also be a combination of these phenomena. It can also be a degradation caused by manipulation of the DNA, for example during its extraction from a cell.

 Preferably, to evaluate the quality of a human DNA, the target gene used is human EGFR1.

 The subject of the present invention is also the use of the in vitro method for analyzing the variation of the quantity of a target gene different from SEQ ID NO: 1 defined above for determining the effect of a substance and / or a treatment on the number of copies of a target gene in a sample.

 The present invention also relates to the use of the in vitro method for analyzing the variation of the amount of a target gene different from SEQ ID NO: 1 defined above for determining the toxicity of a substance and / or of a treatment on the number of copies of a target gene in a sample.

In the present context, the term "effect of a treatment on the number of copies of a target gene" means the effect caused by physical and / or biological and / or chemical phenomena on the number of copies of the target gene. For example, physical phenomena can be a UV irradiation of the DNAs, prior lyophilization of the tissues, heating of the DNAs, etc.

 The present invention also relates to the use of the in vitro method for analyzing the variation of the amount of a target gene different from SEQ ID NO: 1 defined above in a screening test.

 For example, the screening test may be a screening test for a substance or a tumor.

 A screening test can, for example, detect the effect of a substance or a treatment on the genome of an individual. The treatment may be, for example, a protective, positive, therapeutic or toxic treatment. For example, the treatment may be selected from the group consisting of UV exposure, irradiation, application of a molecule. For example, the molecule may be chosen from the group comprising anti-UV agents, antioxidants, antimutagens, antitoxics and protectors of DNA and its replication.

 The subject of the present invention is also the use of the in vitro method for analyzing the variation of the amount of a target gene different from SEQ ID NO: 1 defined above to help determine and / or adapt a treatment and / or follow the evolution of a disease during its treatment.

 The present invention also relates to a human genomic DNA analysis kit comprising sense and antisense primers for amplifying the sequence SEQ ID NO: 1 of the calibrator, and a fluorescent probe for quantifying the amplicons of SEQ ID NO : 1 products.

 For example, the sense and antisense primers for amplifying the sequence SEQ ID NO: 1 of the calibrator can be respectively of SEQ ID NO: 2 and SEQ ID NO: 3 sequences.

 The fluorescent probe making it possible to quantify the amplicons of SEQ ID NO: 1, products can be for example of sequence SEQ ID NO:

4. This fluorescent probe to quantify the amplicons of SEQ ID NO: 1, products may be, for example, a probe of sequence SEQ ID NO: 4.

The inventors are the first to have identified that the human EGFR1 gene (chr 7: 55224226-55238906) can be used to evaluate the quality of a human DNA.

 They are also the first to have identified a simple and quantitative method for quantifying the genomic DNA of a gene involved in pathologies. For example, genes involved in pathologies may be selected from the group consisting of human EGFR1, human EGFR2, human EGFR3, human EGFR4, human C-ERB-B2, human ERBB3, human ERBB4, human TOP I, human TOPO 2 ALPHA, Human MDR, human MET, human IGFR1, human SDMAD4, human TNFRSF10A (TRAIL R4), TNFRSF10B (TRAIL-R2 - CDC262), human TPBG, and human VEGR2-KDR-FLK1. This method can be used to better predict and monitor the effectiveness of a treatment using for example a targeted treatment, for example a monoclonal antibody, acting on one or the other gene and to evaluate the possible resistance to therapies, for example, targeted therapies or chemotherapies.

 They have also identified that the use of a combination of these target genes can be used. It may for example be the combination of the target genes selected from the group comprising MITOC / Genotox presented in Table 1 above, to evaluate over time the toxic and mutagenic effects of substances administered, in particular the substances administered at very high doses. low doses over long periods in in vivo or in vitro model systems (reconstituted tissue explants, model cells, tissue or organ models or even animal models).

As used herein, the term "MITOC gene" means the gene amplified by the sense and antisense sequences SEQ ID NO: 66 and SEQ ID NO: 67. They are also the first to have identified that target genes selected from the group consisting of MITOC, human CDKN2-P16 (or human CDK4-P16), human ADAMTS18, human LRP1 B, human GRID2 (or human GRIDC4), human FHIT, SPOPL human, human MMRN1, human AGAP2, human CNTNAP4 and human FNLB, for example in the group comprising MITOC, human CDKN2-P16, human ADAMTS18, human LRP1 B, human GRIDC4 and human FHIT, can be used alone or in combination to demonstrate possible chronic toxicity and / or genotoxicity induced by a given substance, by examining changes in the mitochondrial genome and chromosomal genome variations, respectively. Preferably, variations of the mitochondrial genome and variations of the chromosomal genome are observed at specific locations. These are, for example, fragile sites of DNA selected from the group comprising LRP1B (chr 12: 57,522,282-5 57,543,841), FHIT (chr 3: 59735421 -61237133), P16 (chr 9:21, 967,752-21, 975,038), ADAMTS18 (chr 16: 77389837-7746901 1), GRID2 (chr4: 93225550-94693648), CDKN2-P16 (chr12: 58,145,312-58,145,253), AGAP2 (chr2: 58,127,033-58,127,141), CNTNAP4 (chrl 6: 76,343,915); - 76,344,061), SPOPL (chr2: 139,265,004-139,265,108), GRIDC4 (chr4: 93226519-93226639), MMRN1 (chr4: 90,819,716-90,819,827) and FLNB (chr3: 57,995,790-57,995,904).

 A target gene chosen for example from the group consisting of human EGFR1, human ERBB3 and human ERBB4 can be used as a negative control.

 The inventors have also identified a nucleic acid sequence of sequence SEQ ID NO: 5. This sequence is, like the nucleic acid of sequence SEQ ID NO: 1 in humans, very conserved in mice.

The nucleic acid SEQ ID NO: 5 may be used for murine genomic DNA analysis. The nucleic acid SEQ ID NO: 5 can be used as a calibrator for murine genomic DNA analysis.

 As used herein, "murine genomic DNA" means the ensemble comprising the murine chromosomal genome and the murine mitochondrial genome.

 The term "murine chromosomal genome" is understood to mean the entire chromosomal material contained in the chromosomes of a mouse.

 The term "murine mitochondrial genome" means all the chromosomal material contained in the mitochondria of a mouse.

 The sequence SEQ ID NO: 5 can be used in an in vitro method of genomic DNA analysis, for example in the process of the invention above in which the sequence SEQ ID NO: 5 replaces the sequence SEQ ID NO : 1, and wherein the target gene different from SEQ ID NO: 5 replaces the target gene different from SEQ ID NO: 1.

 This sequence can also be used to evaluate the quality of murine DNA.

 It can also be used to determine the effect of a substance or a treatment on the number of copies of a target gene in a sample.

 It can also be used to determine the toxicity of a substance or a treatment to the number of copies of a target gene in a sample.

 It can also be used in a screening test. This screening may be for example a screening of substances or tumors.

 It can also be used to help determine and / or adapt a treatment and / or monitor the progress of a disease during its treatment.

The terms "DNA quality", "Effect of a substance on the number of copies of a target gene in a sample" and "Substance Screening Test" have the same definition as the one presented above, but reported with the mouse. The different tissues of the mouse can then be analyzed with DNAqual, MITOC and Genotox methods presented above.

 A murine genomic DNA assay kit comprising sense and antisense primers for amplifying the SEQ ID NO: 5 sequence of the calibrator, and a fluorescent probe for quantifying the amplicons of SEQ ID NO: 5 produced, was also developed by the inventors.

 In this kit, the sense and antisense primers for amplifying the sequence SEQ ID NO: 5 of the calibrator can be, for example, respectively of SEQ ID NO: 6 and SEQ ID NO: 7 sequences.

 The fluorescent probe making it possible to quantify the amplicons of SEQ ID NO: 5, produced can be for example of sequence SEQ ID NO: 8. This fluorescent probe making it possible to quantify the amplicons of SEQ ID NO: 5, products can be for example a probe of sequence SEQ ID NO: 8.

 In addition, the inventors have developed sense and antisense primers, as well as fluorescent probes specific for each of these target genes. These target genes, sense and antisense primers and fluorescent probes are shown in Table 2 below. In this table, there is also an example of the sequences of sense and antisense primers and of a fluorescent probe specific for the nucleic acid of sequence SEQ ID NO: 5.

Table 2: Possible applications in function of the target genes

 analyzes

 Primer primer

 Probe

Uses Target genes antisense sense

 SEQ ID NO:

 DNAqual EGFR1 murine 69 70 71

 CDKN2-P16 murine 72 73 74

ADAMTS18 murine 75 76 77

GENOTOX

LRP1 B murine 78 79 80 Mice

 Murine GRID2 81 82 83

Murine FHIT 84 85 86 MITOC Mouse - 87 88 89

The inventors are also the first to have identified that the murine EGFR1 gene can be used to evaluate the quality of murine DNA.

 They are also the first to have identified that a target gene selected from the group consisting of murine CDKN2-P16, murine ADAMTS18, murine LRP1 B, murine GRID2 and murine FHIT, can be used to show the genotoxicity induced by a specific molecule. The murine EGFR1 target gene can be used as a negative control. In combination with the MITOC method, it is also possible to analyze the effects of chronic intoxication and / or genotoxicity of a treatment or a specific substance directly on the animal or in model cells. Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the appended figures, given for illustrative and non-limiting purposes.

Brief description of the figures

FIG. 1 represents a standard dilution curve of human reference laboratory DNA generated by a CFX96 (Biorad) thermocycler with CFX Manager software. "A" represents the slope obtained with a Yakima yellow fluorochrome-labeled probe for the EGFR1 target gene with the following parameters: E = 97.0%, R2 = 0.994, slope = 3.366. "B" represents the slope obtained by the calibrator marked with a Texas Red fluorochrome with the following parameters: E = 93.2%, R2 = 0.999, slope = 3.497. "Ct" means "Cycle threshold", "One" means "unknown", "Std" means "standard" and "Log DNA (i)" means "logarithm of the initial amount of DNA". FIG. 2 represents an automated histogram obtained by a CFX96 (Biorad) thermocycler with the CFX Manager software for a DNAqual test that makes it possible to compare DNA samples before and after UV irradiation. The abscissa is the name of the DNA samples. On the ordinate is the number of copies of the EGFR1 target gene in the different samples. This number is expressed "in number of times" of the value obtained for the reference DNA of the laboratory (50 ng of each). The results are normalized by the universal calibrator RXRbeta, and by comparing each time the crude DNA (50ng) and the same DNA after irradiation with UV (50 ng) represented respectively from left to right: 05, 05UV, 06, 06UV, 07, 07UV, 08, 08UV, 09, 09UV, 1 1, 1 1 UV, 12, 12UV, 13, 13UV, 14, 14UV, 16, 16UV, 18, 18UV, 19, 19UV, 20, 20UV, 21, 21 UV, 22, 22UV, 23, 23UV, 26, 26UV, 27, 27UV, 31, 31 UV, 33, 33UV, 35, 35UV, 40, 40UV, 41, 41 UV, 43, 43UV, 44, 44UV, 46, 46UV, 48, 48UV, 49, 49UV, 50, 50UV, 57, 57UV, 62, 62UV, 64, 64UV, STD100, STD50 and STD25. Each number presented in this figure corresponds to samples treated or not with ultraviolet. "UV" is indicated next to this number when the DNA sample has been treated with ultraviolet light. STD100, STD50, and STD25 represent dilutions of a laboratory reference DNA (100 ng, 50 ng, and 25 ng, respectively) that serve for this control DNA experiment to express the results. They make it possible to check the good efficiency of each duplex qPCR using RXRbeta as normalization calibrator.

FIG. 3A represents a photograph of the result of electrophoresis of human DNAs (2 μl of each duplicate) on 0.8% agarose gel before UV irradiation. The numbers have the same meaning as for FIG. 2. FIG. 3B represents a photograph of the result of electrophoresis of human DNAs (2 μl of each duplicate) on 0.8% agarose gel after UV irradiation of 120 mJoules. / cm2 for 15 min in the UV device Stratalinker 1800 (Stratagene) (catalog: No. 400072). The numbers have the same meaning as in Figure 2. "M" means "molecular weight marker".

 FIG. 4 represents a histogram obtained by a CFX96 (Biorad) thermocycler with the CFX manager Software for a DNAqual mouse test (50 ng) carried out on DNA duplicates extracted from freeze-dried tissues with respect to the DNA extracted from the same fresh tissues not freeze dried. This histogram represents on the abscissa: the name of the samples and on the ordinate: the number of intact copies of the EGFR1 gene, normalized by the RXR beta calibrator and with respect to the number of intact copies of the EGFRI, measured in the DNA extracted from fresh tissues . C2 represents the DNA extracted from fresh tissues, E1, E2, E3 and E4 respectively represent DNA extracted 24 hours, 1 week, 1 month and 3 months after their lyophilization (duplicates of C2). STD100, STD50 and STD25 represent respectively mouse reference DNA dilutions of: 100 ng, 50 ng and 25 ng which verify the good efficiency of duplex qPCR.

 FIG. 5 represents a photograph of the result of an electrophoresis of a duplicate sample of 100 ng of these same mouse DNAs on agarose gel. C2, E1, E2, E3 and E4 have the same meaning as for Figure 4. "M" means "molecular weight marker", "C1" means respectively "control 1" or a duplicate of C2, "A", " B "and" C "respectively mean" laboratory internal standards corresponding to non-degraded, reference murine murine ".

FIG. 6 represents a histogram obtained with an Excel spreadsheet for tests: MTT, DNAqual and MITOC (represented by the stick triplets respectively from left to right). The MTT result represents the percentage inhibition of the proliferation of HEPG2 cells equipped enzymatically with cytochrome P450, as a function of their intoxication for 72 hours with substances numbered 38, 133, 145, 158, 167, 170, 194, 198, 203, 21, 248 and 251 (all were diluted in DMSO and then in culture medium at the final concentration of 15 μΜ). "DMSO" means "dimethylsulfoxide" and "DMSO +" corresponds to cells treated with 0.01% DMSO. The MITOC and DNAqual tests were performed on the DNA extracted from the treated cells, after the MTT test, and their results were taken from the automated histograms obtained following the same procedure as that described previously. The control DNA is "DMSO +".

 FIG. 7 represents a histogram obtained with an Excel spreadsheet for a Copycount test on tumor / healthy tissue pairs taken from 10 patients (represented by the doublets of sticks respectively from left to right). The abscissa represents the number of the patient (1 to 10) with the target gene used (EGFR1, IGFR1 or ERBB3). The ordinate represents the relative expression for each target gene normalized by the calibrator gene.

 FIG. 8 represents a histogram obtained with an Excel spreadsheet for a MITOC test on treated HepG2 cells (with 100 nM orange Acridine (FIG. 8A) or with 100 nM methyl methanesulfonate (MMS) (FIG. 8B)) . The abscissa represents the sampling days from the treatment (D3, D6, D10, D13, D17 and D20). The ordinate represents the relative expression (in percentage) for MITOC normalized by the calibrator gene.

EXAMPLES

Example 1: Material and Method:

 The experiments presented in the examples below were carried out using the principle of duplex qPCR.

They were carried out using a CFX96 thermocycler (Biorad) using CFX Manager software. Extraction of the DNA from these cells was performed according to the protocol described in Sambrook J, Russel D. Molecular Cloning: A Laboratory Manual. 3rd edition. Flight. 3. New York, NY, USA: Cold Spring Harbor Laboratory Press; 2001 [6].

 The qPCR was carried out on 96-well plates "hard-shell 96-

Well Thin-Wall PCR Plates "by Biorad.

 Reaction mixtures were prepared according to Table 3 below.

Table 3: PCR mix preparation

The primers and probes of the target gene are specified in each of the examples below. Similarly, the amount of DNA sample and water used in the reaction mixture is specified below.

 Triplicates were made for each of the measurements.

 Each well of the 96-well plate for qPCR was filled with a final reaction volume of 15 μί.

 The thermal cycler has been programmed to obtain the following amplification steps:

 - initial denaturation:

 o 3 min at 95 ° C.

- PCR cycles (x 40): o 10 seconds at 95 ° C;

 o 20 seconds at 63 ° C;

 o 30 seconds at 72 ° C (+ data collection and analysis at this stage of the cycle).

 The reference gene, that is to say the calibrator according to the invention, was amplified using primers sense sequence SEQ ID NO: 2 and antisense sequence SEQ ID NO: 3. The amplicons produced were quantified using a probe of sequence SEQ ID NO: 4 comprising a fluorochrome 1, here the Texas red (registered trademark). The qPCR software is told that this fluorochrome 1 is the calibrator ("cal").

 The target gene was amplified using specific sense and antisense primers and defined in each of the examples below. The amplicons produced were then quantified using a probe comprising a fluorochrome 2, here Yakima yellow (registered trademark). The qPCR software is told that this fluorochrome 2 is the target ("target").

A range corresponding to a DNA reference dilution cascade ranging from 25 ng to 100 ng was performed and also served as a control sample to express the results. Each point of this range is made in triplicate. This range allowed to control the parameters of the qPCR.

The thresholds and thresholds of each fluorophore were manually adjusted to obtain the best known dilution curve of reference DNA: presenting optimum PCR efficiency parameters (slope, efficiency and R ² ) and differences in amplification efficiency included. between 0 and 5% for the given target gene and for the calibrator gene. These constants of and were taken up for the interpretation of the results of all subsequent experiments concerning this given target gene.

Without this dilution range, the qPCR software does not show us the efficiency of the two amplifications, nor their slope, nor the correlation coefficient of Pearson R ² . Indeed, this software is made for measure a standard curve of cDNA cascade dilutions and make the relative target cDNA copy assay relative to a reference cDNA.

 The standard genomic DNA dilution curve was generated by the thermocycler from the standard DNA dilution range with the "Ct obtained" on the ordinate and the log of the DNA concentration of the reference sample. On the abscissa. This standard curve is shown in FIG. The results obtained with the samples to be tested are indicated by XXX. They are positioned relative to references 25, 50 and 100 ng and are each expected to contain 50 ng of DNA. The software will calculate how much DNA each sample to be tested actually contains. We then interpret it as a number of copies of genomic DNA relative to the number of calibrator and target gene copies indicated for the control DNA of the experiment which is here also the reference DNA.

The linear regression equation with the Pearson R ² correlation coefficient showed a very good correlation between Ct values and the amount of DNA present in the reference samples (dilution range).

 The standard curve obtained by analyzing the dilutions of the reference DNA made it possible to affirm that all the selected qPCR conditions are adequate for analyzing the variation in the number of copies of a target gene. For this purpose, the qPCRs were carried out on 96-well plates and with reaction mixtures still comprising the DNA range (reference: 25 ng, 50 ng and 100 ng), while retaining the same Ct plate parameters. plate.

In DNA to be tested, if there is gene amplification of the target gene, the yellow probe leaves before orange (early Ct), that is to say, after application of the AACt method and conversion to 2 ^_ΔΔε , the value obtained for this "ratio of fluorescences" is greater than 1 and greater than that obtained for the reference DNA. This ratio can reach 10 see more. It is the opposite if there is a deletion (delayed Ct).

 In both cases, the Ct of the calibrator makes it possible to correctly "locate" the DNA on the dilution range of the reference DNAs and to find that there is no lack of calibrator matrix DNA when compared to DNA. reference (Ct <28).

 If there is a joint decrease in the number of intact copies and the target gene and the calibrator, the ratio does not change but the amount of DNA is calculated by the software as less than that indicated relative to the reference DNAs. It translates here a lack of matrix DNA intact and signs for us a degradation of the tested DNA, compared to the reference DNA (measurement of 50 ng of DNA in all cases).

 The measurements obtained for different samples to be tested may appear on an automated histogram as a number of times in addition to or less than the number of copies (X) of target genes observed for the tested sample relative to zero. A variant of the software makes it possible to choose, in addition, a DNA sample as an internal reference and the results can then be in the form of a different histogram, the zero being this sample.

This method therefore makes it possible to evaluate the quality / integrity of the DNA by evaluating the number of intact matrix copies present in the samples to be tested by comparison with the normal number of matrix copies present in the reference DNA. In the examples below, similar amplification efficiencies for the two genes (calibrator and target), namely between 90 and 1 10%, associated with linear correlations (R ² )> 0.9, as well as a wide range of detection, it was possible to determine in 25 to 50 ng of the DNA sample to be analyzed (test sample), the number of copies of the target gene by the Ct ΔΔ method, also called the "method of

Livak ". Example 2 DNAqual - Quantification of the Human EGFR1 Gene According to the Method of the Invention on DNAs Having Undergone Artificial Degradation by a Physical Treatment or UV Irradiation

In this example, the DNA to be analyzed came from lung cells (Lyon Biological Resource Center). Duplicates of each sample were or were not subjected to UV irradiation of 120 mJ / cm ² for 15 minutes using a UV Stratalinker 1800 (Stratagene Catalog No .: 400072) before being analyzed by DNAqual and also by agarose gel electrophoresis.

 The experiment was carried out on 32 DNA samples to be analyzed, extracted from the aforementioned cells. They are denoted respectively: 05, 06, 07, 08, 09, 1 1, 12, 13, 14, 16, 18, 19, 20, 21, 22, 23, 26, 27, 31, 33, 35, 40, 41, 43, 44, 46, 48, 49, 50, 57, 62 and 64, and correspond to extractions of DNA from different tissue samples.

 Samples STD100, STD50 and STD25 correspond to dilutions of the range of laboratory reference DNA (100 ng, 50 ng and 25 ng, respectively) that did not undergo UV irradiation. The control DNA used to express the results is the same as the reference DNA (50 ng)

 The samples named with a suffix "UV" correspond to the irradiated samples. When this suffix is not indicated, it is the paired control samples, that is to say, not having been irradiated by UV. For each of these samples, 50 ng was analyzed.

 The reagents used for qPCR were as follows:

 - iQ supermix (trademark) (reference 1708862, Biorad),

- primer direction of the calibrator (SEQ ID NO: 2) to 10 μΜ,

- antisense primer of the calibrator (SEQ ID NO: 3) at 10 μΜ, calibrator probe (SEQ ID NO: 4) having a 5 'fluorochrome Texas red (registered trademark) and a 3', 20 μΜ quencher BHQ2 (registered trademark)

 sense primer of EGFRI (SEQ ID NO: 9) at 10 μΜ,

 EGFRI antisense primer (SEQ ID NO: 10) at 10 μM,

 EGFR1 probe (SEQ ID NO: 1 1) having a Yakima yellow fluorochrome (registered trademark) in 5 'and a BHQ1 quencher (registered trademark) in 3', at 20 μM.

 All primers and probes were provided by Eurogenetec.

In this example, the number of copies of a target gene (human GFR1) was quantified in comparison with the number of intact copies of the calibrator according to the invention of sequence SEQ ID NO: 1 present in any DNA sample. human.

 In an individual to be tested, the sample was previously quantified by measuring the optical density of the sample, ie 50 ng / μί of DNA.

 The same measurement was performed on a reference DNA sample which contains a reduced copy number of human EGFRI and a reduced number of copies of the RXR beta calibrator (SEQ ID NO: 1) due to their UV degradation.

 The two genes, RXRbeta (SEQ ID NO: 1) and EGFR1, were measured as synthesized amplicons during a duplex real-time qPCR. The sequence of the calibrator was amplified using the primers sense SEQ ID NO: 2 and antisense SEQ ID NO: 3. The sequence of the EGFR1 gene was amplified using primers sense SEQ ID NO: 9 and antisense SEQ ID NO: 10.

 The amplicons produced were detected by their hybridization with the two specific probes described above, which were put in duplex. The analysis software was programmed to detect the Texas Red branded calibrator probe as a "calibrator", and the probe

EGFR1 as "target" and reference DNA as control. For a reference DNA for the human EGFR1 gene, the number of intact copies of the human EGFR1 gene is identical to that of the RXR beta gene, namely 660 diploid copies per ng of DNA (ie 330 haploid copies). Thus, in a reference individual, the number of copies of GFR1 and RXRbeta varies from 1 +/- 5%. Thus the number of copies of the human EGFR1 gene has theoretically decreased.

 Sample STD100 was chosen as a reference sample. It has been noted indicated as "control or standard" in the thermocycler program.

 All results were expressed relative to the STD100 reference DNA (UV irradiated) of the laboratory.

The overall results are shown in Figure 2 and in the table below.

Table 4: Experimental Results of the DNAqual Test in Humans

Target Human Caliper EGFR1 Human Expression

 Ech. Mean C (t) C (t) SEM Avg C (t) C (t) SEM Exp. Exp. SEM

5 27.4 0.02318 26.24 0.01309 3.66924 0.06889

05 UV 27.36 0.07419 28.5 0.05694 0.72107 0.0476

 6 26.98 0.03062 25.35 0.01895 5.15549 0.13094

06 UV 27.39 0.03225 28.63 0.02866 0.6741 0.02054

7 28.1 0.02744 26.49 0.02644 5.04394 0.13581

07 UV 28.54 0.086 29.78 0.05445 0.66341 0.04764

8 27.74 0.1677 25.5 0.09182 7.91913 0.96682

08 UV 28.07 0.04776 29.44 0.08605 0.60569 0.04221

9 28.35 0.04453 26.49 0.06748 6.03317 0.34523

09 UV 28.27 0.03126 29.64 0.0597 0.60653 0.02895

11 26.85 0.00843 25.01 0.01452 5.96881 0.07094

11 UV 27.43 0.07957 28.68 0.06745 0.66162 0.04874

12 27.76 0.05285 25.9 0.07374 6.05175 0.38848

12 UV 28.38 0.07031 29.73 0.12071 0.6192 0.06124

13 27.06 0.05981 25.55 0.03257 4.72951 0.22712

13 UV 27.92 0.0199 29.2 0.01981 0.64547 0.01281

14 27.87 0.04584 25.98 0.10219 6.16115 0.48888

14 UV 28.45 0.06608 29.85 0.07029 0.59274 0.04042 6 27.35 0.01609 25.82 0.01787 4.77717 0.08122 UV 28.05 0.06742 29.34 0.06433 0.64383 0.042398 27.3 0.04712 25.91 0.09067 4.34503 0.31444 UV 27.91 0.06075 29.16 0.03099 0.66374 0.031919 28.08 0.06157 25.96 0.05055 7.22236 0.40525 UV 28.54 0.04343 30.08 0.04173 0.53628 0.022820 27.41 0.04159 26.02 0.0769 4.33398 0.26833 UV 28.23 0.02581 29.63 0.05687 0.59377 0.026271 27 , 78, 0, 1484, 25.88, 0.26152, 6.20689, 32459, UV 27.98, 0.04963, 29.34, 0.01098, 0.61037, 0.021842, 27.3, 0.07329, 25.74, 0.0705, 88582 0.35103 UV 27.89 0.03789 29.12 0.03966 0.66954 0.025963 26.82 0.0946 25.39 0.09151 4.46986 0.41567 UV 27.71 0.06397 29 05 0.00393 0.62312 0.0281 16 26.05 0.12279 24.42 0.08825 5.19679 0.5546 UV 24.8 0.05276 25.92 0.05275 0.73952 0.038997 26, 6 0.03057 25.04 0.04926 4.91212 0.20158 UV 25.97 0.05777 27.24 0.06443 0.66176 0.040481 26.25 0.0571 1 25.02 0.1 1391 3 , 8909 0.351 18 UV 25.84 0.01954 27.08 0.01003 0.67491 0.010453 25.99 0.1880 24.46 0.04542 4.81953 0.49348 UV 25.39 0.09012 26, 51 0.08633 0.73895 0,065155 26,34 0,13858 24,76 0,13886 5,00667 0,69406 UV 25,06 0.04449 25.97 0.01718 0.85625 0.028770 26.45 0.06492 25.1 0.06322 4.19974 0.2689 UV 25.16 0.01 152 26.17 0.02433 0.80007 0.015261 26.45 0.06258 25.1 1 0.14908 4.21165 0.48252 UV 26 , 69 0.04885 27.87 0.09019 0.70053 0.050883 27.64 0.13903 26.33 0.0677 4.09253 0.44613 UV 26.9 0.0581 1 27.94 0.05533 0 , 7787 0.044144 27.78 0.14671 25.48 0.14336 8.24431 1, 19486 UV 27.28 0.1018 28.48 0.05606 0.69179 0.058566 26.65 0.07212 25, 0.01719 3.9091 0.20408 UV 26.84 0.03251 28.1 0.03454 0.66414 0.022268 26.16 0.01007 25.07 0.04093 3.5274 0.10544 UV 25 78 0.02089 27.03 0.06463 0.66811 0.032179 26.72 0.04256 24.77 0.07539 6.48324 0.39744 UV 27.03 0.05727 28.17 0.04712 0.72097 0.037750 25.21 0.04301 24.34 0.00644 3.0359 0.09294 UV 25.25 0.02312 26.33 0.01546 0.75892 0.014897 25.7 0.02297 25.24 0 , 06377 2.26053 0.10859 57 UV 25.38 0.04469 26.51 0.0644 0.73659 0.04086

62 27.44 0.05574 26.34 0.04313 3.51752 0.17501

62 UV 27.63 0.03988 28.95 0.10344 0.63199 0.04965

64 26.32 0.02564 25.3 0.00947 3.34445 0.0644

 64 UV 27.42 0.06883 28.25 0.05031 0.89459 0.05383

STD100 23.62 0.0687 24.33 0.06883 1 0.06872

STD25 25.6 0.04084 26.29 0.03063 1, 00333 0.03615

STD50 24.69 0.06741 25.4 0.05195 0.98873 0.0594

 In this table "Ech. "Means" Sample "," Average Ct "means" average Ct "," SEM "means" standard error "and" Exp. "Means" expression ".

The value corresponding to sample STD100 is 1 because it is the reference sample, marked as standard in the qPCR program.

 The values corresponding to samples STD50 and STD25 are almost equal to 1, which is normal because the dilution does not change the fluorescence ratio of the calibrator and the human EGFR1 gene.

 The values corresponding to the UV non-irradiated samples (05, 06, 07, ...) are all greater than 1 and all higher than the values corresponding to the samples irradiated by UV (respectively 05UV, 06UV, 07UV, ...). This means that the number of copies of the human EGFR1 gene is higher before the UV treatment. There is usually 3 times more matrix DNA before irradiation than after. It should be noted that standard DNA is degraded by UV in the same proportions.

 These results show a clear degradation of the UV-exposed DNAs which reaches 60% of the non-irradiated controls on average

 The method according to the invention makes it possible to effectively detect and quantify the variation in the number of intact and therefore amplifiable copies by qPCR of the EGFR1 gene, caused by a UV treatment.

A study of the variation in the number of copies of the EGFR1 gene with DNAs extracted from the abovementioned cells and under the same conditions procedures as those described above, was performed with an agarose gel analysis method. 6 μl of each DNA sample, before and after irradiation, were deposited on a 0.8% agarose gel. The molecular weight marker used was Eurogenetec's Smart Ladder: MW-1700-10.

 The results obtained and presented in FIGS. 3A and 3B clearly demonstrate that this method does not make it possible to quantify the number of intact copies of the EGFR1 gene before and after UV treatment. Example 3 DNAqual - Quantification Kit of the Human EGFR1 Gene

A kit for quantitation of the EGFR1 gene has been made in and comprises the following components described in Example 2 above:

10 μΙ of reference DNA for EGFR1 at 100 ng / μl,

 10 μΙ of reference DNA for EGFR1 at 50 ng / μl,

 10 μΙ of reference DNA for EGFR1 at 25 ng / μl,

 - 770 μΙ of iQ supermix (trademark) (reference 1708862, Biorad),

 - 50 μΙ of primer direction of the calibrator (SEQ ID NO: 2) at 10 μΜ,

 - 50 μΙ of antisense primer of the calibrator (SEQ ID NO: 3) at 10 μΜ, - 16.5 μl of calibrator probe (SEQ ID NO: 4) having a 5 'Texas red fluorochrome (registered trademark) and a BHQ2 quencher (registered trademark) in 3 ', at 20μΜ,

 - 50 μΙ sense primer EGFRI (SEQ ID NO: 9) at 10 μΜ,

 50 μl of EGFRI antisense primer (SEQ ID NO: 10) at 10 μΜ,

 16.5 μl of EGFR1 probe (SEQ ID NO: 1 1) possessing a Yakima yellow fluorochrome (registered trademark) at 5 'and a BHQ1 quencher (registered trademark) at 3', at 20μ.

Each of these components is separately packaged in a plastic tube of 1.5 ml (Dutscher, catalog no .: 045401). Example 4 DNAqual - Quantification of the murine EGFR1 gene according to the method of the invention on tissues of mice having undergone or not lyophilization before the purification of their DNAs

 The experiment described in Example 2 was performed on DNAs from mouse cells / tissues shown in Table 5 below.

 This example was carried out using the sequences SEQ ID Nos. 6 and 7 as sense and antisense primer, and the sequence SEQ ID NO: 8 as a probe for amplifying and detecting the universal calibrator in the mouse.

 The sequences SEQ ID Nos. 69 and 70 as sense and antisense primer, and the sequence SEQ ID NO: 71 as a probe for amplifying and detecting the murine EGFR1 target gene. Table 5: Cells / Tissues Used

 Number of

 Characteristic of the sample the sample

 DNA extracted from mouse liver at the time

C2 3 months but frozen at time T0 =

 positive control

 DNA extracted from mouse liver, freeze-dried

E1 at time T0, but purified 24 hours

 after freeze-drying

 DNA extracted from mouse liver, freeze-dried

E2 at time T0, but purified 1 week

 after freeze-drying

 DNA extracted from mouse liver, freeze-dried

E3 at time T0, but purified 1 month after

 lyophilization

 DNA extracted from mouse liver, freeze-dried

E4 at time T0, but purified 3 months later

 lyophilization

 Reference DNA mouse at 100 ng

 STD100

 (not freeze-dried)

 Mouse reference DNA at 50 ng

 STD25

 (not freeze-dried)

 Mouse reference DNA at 25 ng

 STD50

(not freeze-dried) Lyophilization of the tissues E1, E2, E3 and E4 was carried out using a benchtop freeze-dryer MARTIN CHRIS of 2.5 kg, - 55 ° C | ALPHA 1 -2 / LDplus.

 The DNA of lyophilized tissues E1, E2, E3 and E4 was extracted respectively 24 hours, 1 week, 1 month and 3 months after lyophilization of these tissues.

The overall results are shown in Figure 4 and Table 6 below.

Table 6: Experimental Results of the DNAqual Test in the Mouse

 In this table "Ech. "Moy Ct", "SEM" and "Exp. Have the same meaning as before. The value corresponding to sample STD100 is 1 because it is the reference sample, marked as standard in the qPCR program.

 The values corresponding to samples STD50 and STD25 are almost equal to 1, which is normal because the dilution does not change the fluorescence ratio of the murine calibrator and the murine EGFR1 gene.

The value corresponding to sample C2 is close to 1, which means that the number of copies of the murine EGFR1 gene is almost identical to that of the reference sample. The values corresponding to the freeze-dried samples (E1, E2, E3 and E4 are all less than 1 and less than the value corresponding to the non-freeze-dried sample, which means that the number of copies of the murine EGFR1 gene is lower after lyophilization.

 These results show that the method according to the invention makes it possible to effectively detect and quantify the variation in the number of copies of the murine EGFR1 gene, caused by lyophilization.

A study of the variation in the number of copies of the EGFR1 gene with DNAs extracted from the aforementioned cells / tissues and under the same operating conditions as those described above, was carried out using an agarose gel analysis method. 6 μl of each DNA sample were deposited on a 0.8% agarose gel. The molecular weight marker used was Eurogenetec's Smart Ladder: MW-1700-10.

The results obtained and presented in FIG. 5 clearly demonstrate that this method does not make it possible to detect the degradation of the DNA generated by prior lyophilization of the tissues. Example 5: MITOC - Verification of the Potential Toxicity of Drug Candidates - Comparison with the Standard MTT Cell Proliferation Assay

 In this example, the reference DNA and the DNA to be analyzed came from HEPG2 model cells equipped enzymatically with cytochrome P450 ("CYP 450").

 The DNA to be analyzed was determined to belong to cells intoxicated with a given substance for 72 hours. The reference DNA was determined to belong to non-intoxicated cells cultured for 72 h in the presence of 0.01% DMSO.

In this example, three tests were performed: an MTT test in order to quantify the viability of the cells treated with possibly anti-proliferative substances,

 - a DNAqual test to check the quality of the DNA and

 a MITOC test for quantifying the number of mitochondrial genomes or mitochondria contained in the HEPG2 cells treated or not with substances which, in addition to their anti-proliferative activity, their possible toxicity is to be determined. The information obtained is compared with the results obtained with the MTT test.

FIG. 6 presents the results of this example 5. Each triplet represents the results of the MTT, DNAqual and MITOC tests respectively for each treatment. For example, the ^1st triplet represents the results of the MTT, DNAqual and MITOC tests for DMSO treatment.

For comparison, all the values obtained with the MTT test were converted to a percentage relative to the indicated control "DMSO +" whose percentage was fixed at 0% (for zero% inhibition of cell proliferation). Thus, in Figure 6, in the 1 ^st triplet showing the results with treatment with DMSO ( "DMSO +"), ¹ stick, which represents the result of MTT assay, is zero for "zero% inhibition of cell proliferation ".

 12 different substances, diluted in DMSO, were tested. They were added to the culture medium of HEPG2 cells which were thus maintained for 72 hours before analysis. For this example, the 12 substances tested are numbered 38,

133, 145, 158, 167, 170, 194, 198, 203, 21, 248, 251. These are nucleoside analogues from the chemotherapy library of the Nice Institute of Chemistry, University of Nice Sophia Antipolis, France. Each of these substances was tested at a concentration of 15 μΜ.

The control was carried out with dimethylsulfoxide ("DMSO") without test substance at a concentration of 0.01%. The MTT test was performed according to the protocol described in Hussain et al. (Hussain et al., "Immunol Methods, 1993 Mar 15; 160 (1): 89-96 [7]). MTT tetrazoline salt ("3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide"), included in the kit Cell Titer 96 Non Radioactive Cell proliferation assay (catalog Part # G4001; PROMEGA, France).

 The MTT test was performed in 96-well plates (Orange scientific: Cat. No. 5530 100) with the kit Cell Titer 96 Non Radioactive Cell proliferation assay (catalog Part # G4001), according to the manufacturer's indications with, at the beginning , 7500 cells / well.

 At the end of the MTT test, the plates were emptied of their liquid and the DNAs were extracted from the well triplicate cells and mixed before being analyzed by DNAqual and MITOC. Their results are expressed in% relative to the results obtained with the control DNA "DMSO +", fixed at 100%.

For the DNAqual test, the same protocol as that described in Example 2 has been implemented. For the MITOC assay, the variation in the number of copies of a mitochondrial target gene was measured according to the protocol described above with the sense and antisense primers of sequences SEQ ID NOs: 66 and 67 respectively, and with a fluorescent probe of sequence SEQ ID 68.

HEPG2 cells were chosen to perform the MITOC test. These are hepatic cells equipped enzymatically with cytochrome P450 ("CYP 450") for the management of toxins. HEPG2 is frequently used to test the toxicity of substances in in vitro tests. The overall results are shown in Figure 6 and Table 7 below. QPCR tests are expressed as% of control.

Table 7: Experimental Results of MITOC and DNAqual Tests in Humans

 In this table "Ech. "Means" Sample "," Prol. "Means" proliferation "," Inh. "Means" inhibition "," SEM "means" standard error "and" Exp. "Means" expression ".

Regarding the MTT test, the results show that the 12 substances tested have an antiproliferative power that varies from control (DMSO only "DMSO +").

This example reveals that substance 251 is the only one that does not have the cell-cellular degradation power of the treated cells (medium stick close to 1) and, at the same time, does not show any variation in the number of cells. mitochondrial genome copies (right stick also close to 1) while inhibiting cell proliferation by 25% (left stick close to 25%). Substances 145, 194, 198, 203 and 248 have similar antiproliferative powers, about 25%, but lead to an increase in the number of mitochondrial genome copies (mitochondrial crisis, right sticks) indicating, a strong metabolic activity of cells or cellular suffering when this figure decreases.

 The substance 133 which has the most important antiproliferative power, of 50%, has pronounced toxic effects since the number of mitochondria or copy of mitochondrial genome decreases strongly (right stick), making fear the appearance of a deleterious toxic effect in case of prolonged administration of this substance. Substance 21 1 causes, at this dose, a strong degradation of the genomic DNA and also a mitochondrial crisis.

 This example shows that the DNAqual and MITOC tests refine the results of a conventional MTT test which only indicates a number of cells surviving substance intoxication without giving any additional indication of the possible mitochondrial toxicity or an effect. elastic on the DNA.

 The MITOC test according to the method of the present invention, refines the results of the MTT test by highlighting the toxic effect of a given substance on cellular mitochondria.

Example 6: Copycount - Screening of human colon tumors

 In this example, the reference DNA comes from the laboratory, it is also the control DNA (50 ng). The DNA to be analyzed comes from tissue samples of patients operated for rectocolic cancer. They are paired each time: healthy tissue removed from the tumor on the operative resection room and tumor tissue. They were provided by the REIMS Regional Hospital Tumor Library, (Patient 1-10, in Table 8 below and Figure 7).

The DNAs were extracted as in the preceding examples and samples of 50 ng of DNA were analyzed in triplicate for to measure the number of genomic copies of human EGFR1, human IGFR1 ("Insulin growth factor receptor 1") and ERBB3 ("epidermal receptor 3") by comparing each healthy tissue and matched tumor tissue for each patient.

 The sense and antisense primer sequences and the sequences of the fluorescent probe used to amplify and quantify the EGFR1, IGFR1 and ERBB3 genes were respectively:

 EGFR1: SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 1 1,

 IGFR1: SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, and - ERBB3: SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

 The sense and antisense primer sequences and the sequence of the fluorescent probe used to amplify and quantify the calibrator according to the invention (SEQ ID NO: 1) were the same as those described above.

 The fluorochromes used to quantify the calibrator (fluorochrome 1) and to quantify the target genes (EGFR1, IGFR1 and ERBB3) (fluorochrome 2) were the same as those described in Example 1.

In this example, the "Copycount" method was used to screen human colon tumors by each time comparing paired tumor samples with their non-tumor tissues (healthy tissue), according to the protocol described in Example 1.

The overall results are shown in Figure 7 and Table 8 below.

Table 8: Experimental Results of the Copycount Test in Humans

 Tumor Colon Healthy Healthy Average Cohort

 Exp. Exp. Exp.

Ech. Name Exp. Expr. Expr.

 SEM SEM SEM

EGFR1 1 Patient 1 2, 1631 0, 148 1, 4606 0.0234 1, 0734 0.0419

EGFR1 2 Patient 2 2.2238 0, 1008 1, 0557 0.0337 EGFR1 3 Patient 3 2,5032 0.0518 1, 0134 0.0399

 EGFR1 4 Patient 4 2,477 0.1036 1, 072 0.0421

 EGFR1 5 Patient 5 1, 9792 0.1288 0.0631

 EGFR1 6 Patient 6 2.3394 0.1 152 1, 1046 0.0226

 EGFR1 7 Patient 7 2.21 0.0276 1, 0446 0.0428

 EGFR1 8 Patient 8 2.9304 0.1455 1, 144 0.0559

 EGFR1 9 Patient 9 2.9933 0.0758 0.8637 0.0318

 EGFR1 10 Patient 10 1, 8494 0.058 0.9757 0.0638

 IGFR1 1 Patient 1 1, 717 0.1655 1, 3597 0.0713

 IGFR1 2 Patient 2 1, 6022 0.0599 0.9354 0.0456

 IGFR1 3 Patient 3 2.4781 0.0836 0.9036 0.0322

 IGFR1 4 Patient 4 1, 5032 0.1235 1, 2716 0.052

 IGFR1 5 Patient 5 1, 7025 0.1351, 0476 0.1246

 1, 023 0.0835

IGFR1 6 Patient 6 1, 7928 0.13131 1, 1332 0.0778

 IGFR1 7 Patient 7 1, 5396 0.08 0.9095 0.0648

 IGFR1 8 Patient 8 2.2002 0.2057 1, 0312 0.2639

 IGFR1 9 Patient 9 2,338 0,1707 0,7756 0,0171

 IGFR1 10 Patient 10 1, 3551 0.1 196 0.863 0.0854

 ERB3 1 Patient 1 1, 625 0.6319 0.7878 0.0406

 ERB3 2 Patient 2 4,495 0,5035 1,0027 0.0508

 ERB3 3 Patient 3 0.2434 0.0171 0.9952 0.0685

 ERB3 4 Patient 4 0.2664 0.0139 1, 003 0.0505

 ERB3 5 Patient 5 0.2095 0.0082 1 0.0864

 0.9815 0.0472

ERB3 6 Patient 6 0.6471 0.0979 1, 0162 0.016

 ERB3 7 Patient 7 0.3828 0.0355 0.8575 0.0303

 ERB3 8 Patient 8 1, 5162 0.1087 0.8682 0.0348

 ERB3 9 Patient 9 4.8464 0.2504 1, 0366 0.0389

 ERB3 10 Patient 10 0.7775 0.1419 1, 2475 0.0549

In this table "Ech. "Means" Sample "," Average Ct "means" average Ct "," SEM "means" standard error "and" Exp. "Means" expression ". It is found that all the healthy tissues have a normal number of copies of each of these receptors (almost equal to 1) and that the tumor tissues all have amplifications of the EGFR1 gene. It can be said when we compare healthy tissues and tissues tumor. With regard to NGFR1, the same observations are possible. In tumors, these are gene amplifications that are not present in the paired healthy tissues of the same subjects. On the other hand, with regard to ERBB3, only patients 1, 2, 8 and 9 have gene amplifications of ERBB3, the others have deletions.

 This example demonstrates the effectiveness of the technology in identifying variations in the number of gene copies in colonic tumors. This Copycount test makes it possible to indicate which specific therapies are the most appropriate and also to evaluate the level of aggressiveness of a tumor. Indeed, gene variations can modulate the aggressiveness of a tumor (more or less rapid growth, resistance to more or less important treatments). They can also affect the response to a therapy. For example, gene amplification suggests an increase in the expression of the corresponding protein. This protein is a good therapeutic target. Therapeutic treatment specifically targeting this protein will potentially be the most effective. For example, a monoclonal antibody against EGFRI should give a good response to treat a cancer expressing strongly EGFR1. However, if ERBB2 is also highly expressed, it is possible that the cancer cell uses the ERBB2 signaling pathway to compensate for the absence of EGFRI, thus rendering the anti-EGFR1 treatment ineffective. In the case of this double amplification of the genes coding for EGFR1 and ERBB2, another treatment, targeting an entirely different cell signaling pathway, should then be preferred.

The universality of the calibrator allows the comparison of all measurements. The test can be automated and performed at high speed: all measurements are done with the same qPCR program. Other genes than those used in this example can be screened. Example 7: Genotox - Determination of HepG2 cell intoxication by various low dose substances

 In this example, the reference DNA and the DNA to be analyzed came from HEPG2 model cells. HEPG2 cells are hepatic cells equipped enzymatically with cytochrome P450 ("CYP 450") for the management of toxins. HEPG2 is frequently used to test the toxicity of substances in in vitro tests.

 The reference DNA corresponds to non-intoxicated cells and serves as a negative control.

 The DNA to be analyzed was determined to belong to constant dose intoxicated cells, ie 100 nM, with a given substance for 20 days.

 The toxic substances used were either orange acridine (CAS 65-61-2, Sigma) or methyl methanesulfonate (MMS) (CAS 66- 27-3, Sigma). Orange acridine is an intercalating agent of DNA whose effects at low doses are unknown. MMS is a DNA methylating agent that produces apurinic sites that is transformed into single-strand breaks during DNA repair because of the base repair / excision system.

 These toxic substances were diluted in RPMI 1650 culture medium (Dutscher, PO4-16500) in the presence of 10% Fetal Calf Serum (Dutscher, 500101), Penicillin (50 units / mL) and Streptomycin (50 μg). / mL) (Dutscher, P06-07050).

 All the experiments were done in duplicate.

Every 3 or 4 days (D3, D6, D10, D13, D17 and D20 after the beginning of the experiment), the cells were detached from the culture support by trypsinization (addition of trypsin in the culture medium). The cells were counted and put back into culture at a constant number, ie 5.10 ⁵ cells / 25 cm ² . At each counting point, 5.10 ⁶ cells were harvested to prepare DNA by Proteinase K digestion (Dutscher, 091553) followed by extraction / purification with Phenol-Chloroform (Sigma, 77617).

 The DNAs were then assayed by absorbance at 260 nm and then diluted to 50 ng / L.

At this dose, no proliferation abnormality was found as indicated by the cell counts which remain identical to the untreated control, ie 2.10 ⁶ cells / 25cm ² .

The sense and antisense primer sequences and the fluorescent probe sequences used to amplify and quantify the LRP1 (Chromosome (Chr) 2), SPOPL (Chr 2), GRIDC4 (Chr 4), MMRN1 (Chr 4), CDK4- P16 (Chr 12), AGAP 2 (Chr 12), ADAMTS 18 (Chr 16), CNTNAP 4 (Chr 16) were respectively:

 LRP1: SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92,

 SPOPL: SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,

 GRIDC4: SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 98,

 MMRN1: SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, CDK4-P16: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104,

- AGAP2: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107,

ADAMTS18: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1,

CNTNAP4: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,

The sense and antisense primer sequences and the sequence of the fluorescent probe used to amplify and quantify the calibrator according to the invention (SEQ ID NO: 1) were the same as those described above.

The fluorochromes used to quantify the calibrator (fluorochrome 1) and to quantify the aforementioned target genes (fluorochrome 2) were the same as those described in Example 1. In this example, the "MITOC" and "Genotox" methods were used.

 For the "MITOC" assay, a standard dose / dilution curve was performed with concentrations of 12.5 ng, 25 ng and 50 ng of DNA, used to check the constants of qPCR.

 The variation in the number of copies of a mitochondrial target gene was measured according to the protocol described above with the sense and antisense primers of sequences SEQ ID NO: 66 and 67, respectively, and with a fluorescent probe of sequence SEQ ID 68.

 The results (average measured for each of the 8 aforementioned genes (RPMI)) are shown in Figure 8 and Table 9 below.

Table 9: Experimental Results of the Mitoc Test in Humans

 In this table "Prel. "Means" Sampling "," SEM "means" standard error "and" Exp. "Means" expression ".

These results show that the intoxication of the cells by the MMS causes a cellular suffering which worsens and stabilizes with the time. The cellular suffering is even more important when the intoxication was performed with Acridine.

The MITOC test is therefore used as an indicator of cellular suffering, even though no abnormality of cell proliferation was observed by counting on ten new generations. cell. It also demonstrates the difference in toxicity of different substances.

For the "Genotox" test, the results are shown in Table 10 below. In this table, the results are expressed as percentages relative to the results obtained from the untreated cells at D3, D6, D10, D13, D17 and D20, respectively. Negative values correspond to deletions. Positive values correspond to amplifications.

Table 10: Summary of Genotox results (%) on HepG2

 intoxicated or not

This table 10 makes it possible to compare the effects of the two toxic substances (orange Acridine and MMS) over time at the various chromosomal loci mentioned above.

At chromosome 2, at the SPOL site, a deleterious effect was observed for both substances. It is observed that this deleterious effect is repaired, in time, almost completely for the MMS, but not for the Acridine. At chromosome 2, at the LRP1 site, we note that the effects of the two toxins are opposite: MMS is deleterious whereas Acridine is amplifying. In addition, the effects of MMS are almost repaired at the end of the experiment, while those of Acridine remain severely persistent.

 At chromosome 4, at the MMRN1 site, the observation made for the gene

LRP1 is almost identical to that observed at this site. The deleterious effect of MMS continues over time by a modest amplification while it is major for acridine.

 At chromosome 4, at the GRID2 site, the deleterious effect is pronounced for both toxicants, with a time lag of a weak amplification for MMS at day 10 and at day 17 for acridine.

 At chromosome 12, the effects observed for the two toxins are similar to the AGAP2 site. The cell manages to repair its fragile site in the case of MMS, but not in the case of acridine.

 At chromosome 12, at the P16 site, deletion repair is effective for MMS, whereas it is not effective for acridine.

 Finally, at chromosome 16, at the site of ADAMTS18, the persistent and early amplifications are observed for both toxic, but orange acridine is once again more amplifying.

 At chromosome 16, at the CNTNAP4 site, we observe early, important and persistent deletions.

 Thus, the genotoxic effects observed at low doses have been highlighted by our results. The mechanism of the induced lesions is different for the two molecules.

 The HepG2 cell is equipped to repair deletions and amplifications but the orange acridine causes more serious and more fixed disturbances of the cellular genome. The MITOC test above also indicated this severity of Acridine intoxication.

The Genotox test, implemented using the calibrator according to the invention (SEQ ID NO: 1), makes it possible to detect the effects of toxic substances at doses one thousand times lower than those used for current tests, such as the Comets test or the micro-nuclei test for which the concentrations of toxic substances analyzed are of the order of 100 μΜ.

List of references

[1] Redon et al. "Global variation in the human genome", Nature, vol. 444, 2006, p. 444-454.

 [2] C A Heid, Stevens J, K Livak J, and Williams P M. Real time quantitative PCR. Genome Res. 1996. 6: 986-994

[3] Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M,

Mueller R, Nolan T, MW Pfaffl, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem.

2009, 55 (4): 61 1 -22.

[4] Weglarz L, Molin I, Orchel A, Parfiniewicz B, Dzierzewicz Z.

 Quantitative analysis of the level of p53 and p21 (WAF1) mRNA in human colon cancer HT-29 cells treated with inositol hexaphosphate. Acta Biochim Pol. 2006; 53 (2): 349-56.

[5] "Relative Quantification: Data Management & Analysis Settings",

Ray Meng, (available online at http://genome.hku.hk/portal/files/GRC/Events/Seminars/2009/20090 512 / relative% 20quantification_data% 20management% 20% 20analys is% 20settings.pdf).

[6] Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual. 3rd edition. Flight. 3. New York, NY, USA: Cold Spring Harbor Laboratory

Press; 2001.

[7] Hussain et al. "A new approach for measuring cytotoxicity using colorimetric assay", J Immunol Methods. 1993 Mar. 15; 160 (1): 89-96.

Claims

1. Nucleic acid with sequence SEQ ID NO: 1.

2. Use of the nucleic acid SEQ ID NO: 1 for human genomic DNA analysis.

Use of the nucleic acid SEQ ID NO: 1 according to claim 2 as a calibrator for human genomic DNA analysis.

4. In vitro method for analyzing the variation of the quantity of a target gene different from SEQ ID NO: 1, comprising the use of a nucleic acid of sequence SEQ ID NO: 1, and a step of comparison of the amount of the target gene in a test sample with the amount of the target gene in a reference sample.

The method of claim 4, wherein the step of comparing the amount of the target gene in a test sample with the amount of the target gene in a reference sample is performed using the ΔΔ method of Ct.

The method of claim 4 or 5, wherein the reference sample and the test sample are from a single individual or two individuals.

The method of any one of claims 4 to 6, wherein the amount of the gene is a copy number of the gene.

8. Use of the method according to any one of claims 4 to 7 for evaluating the quality of a human DNA.

9. Use of the method according to any one of claims 4 to 7 for determining the effect of a substance and / or a treatment on the copy number of a target gene in a sample.

The use of the method according to any one of claims 4 to 7 for determining the toxicity of a substance and / or a treatment on the copy number of a target gene in a sample.

11. Use of the method according to any one of claims 4 to 7, in a screening test.

12. Use of the method according to any one of claims 4 to 7, to help determine and / or adapt a treatment and / or follow the evolution of a disease during its treatment.

13. Kit for human genomic DNA analysis comprising sense and antisense primers for amplifying the sequence SEQ ID NO: 1 of the calibrator, and a fluorescent probe for quantifying the amplicons of SEQ ID NO: 1 produced.

14. Kit according to claim 13, wherein the sense and antisense primers for amplifying the sequence SEQ ID NO: 1 of the calibrator are respectively of SEQ ID NO: 2 and SEQ ID NO: 3, and the fluorescent probe for quantifying the amplicons of SEQ ID NO: 1 products is of sequence SEQ ID NO: 4.