Method for the diagnosis of genetic diseases by molecular combing and diagnostic kit

Abstract

The invention concerns a method for detecting or locating one or several genes of one or several specific A DNA sequence or one or several molecules reacting with DNA on a B DNA characterized in that it consists in. (a) fixing and combing a certain amount of said B DNA on a combing surface; (b) reacting the product of the B combing with one or several probes, linked with the gene(s) or specific A DNA sequences. or with the molecules capable of reacting with DNA; (c) extracting information corresponding to at least one of the following categories: (1) the position of the probes. (2) the distance between the probes, (3) the size of the probes (the total sum of size quantifying the number of hybridised probes) for determining there from the presence, the location and/or the amount of genes or specific A DNA sequences. This method can be used in particular for the diagnosis of genetic diseases.

1 . Method for detecting the presence or the location of one or more genes or of one or more sequences of specific A DNA or of one or more molecules reacting with the DNA on a B DNA, characterized in that: 
 (a) a certain quantity of said B DNA is attached to and combed on a combing surface,    (b) the B combing product is reacted with one or more labeled probes, bound to the gene(s) or to the sequences of specific A DNA(s) or to the molecules capable of reacting with the DNA,    (c) the information corresponding to at least one of the following categories is extracted: 
 (1) the position of the probes,  
 (2) the distance between probes,  
 (3) the size of the probes (the total sum of the sizes which make it possible to quantify the number of hybridized probes)  
 so as to deduce therefrom the presence, the location and/or the quantity of the genes or of the sequences of specific A DNA.  
   
     
     
         2 . Method according to  claim 1 , for identifying a genetic abnormality of a break in a genome, characterized in that: 
 (a) a certain quantity of said genome is attached to and combed on a combing surface,    (b) the combing product is hybridized with one or more specific probes corresponding to the genomic sequence for which the abnormality is sought,    (c) the size of the fragments corresponding to the hybridization signals is measured, and    (d) the presence of a break is deduced therefrom either by direct measurement or by comparison with a standard control corresponding to a control length.    
     
     
         3 . Method according to either of claims  1  or  2 , characterized in that the combing product is reacted with one or more labeled probes.  
     
     
         4 . Method according to either of claims  2  or  3 , characterized in that the probe covers a portion of the genome not involved in the break.  
     
     
         5 . Method according to one of  claims 2  to  4 , characterized in that a histogram of the lengths of the probes measured is established.  
     
     
         6 . Method according to  claim 5 , characterized in that the histogram of the lengths of the probes is compared with a histogram produced on a control genome with similar probes.  
     
     
         7 . Method according to  claim 1 , for assaying a given genomic sequence in a genone, characterized in that: 
 (a) a certain quantity said genome is attached to and combed on a combing surface,    (b) the combing product is hybridized with a labeled control probe of length  1 t corresponding to a so-called control genomic sequence, that is to say corresponding to the genomic sequence to be assayed, it will be possible for said probes to be identified separately,    (c) the total length of the hybridization. signals for the two probes, that is to say Lc and Lt, is then measured,    (d) the copy number of the corresponding sequence is calculated for each by the ratio              N                 t     =           L                 t       l                 t                     a                 n                 d                                N                 c     =       L                 c       l                 c                           and the copy number of the sequence to be assayed relative to the control sequence is deduced therefrom.    
     
     
         8 . Method according to  claim 7 , characterized in that the so-called control genomic sequence has a sequence present in a whole number of copies per genome and the presence of a gene abnormality is deduced therefrom when Nc is significantly different from the value expected for the number of genomes present which is deduced from Nt.  
     
     
         9 . Method according to  claim 7 , characterized in that the so-called control genomic sequence consists of various separate portions whose total length  1 t per genome is known, and in that the sequence to be assayed consists of various separate portions whose total length  1 c per normal gene is known, and where the presence of a gene abnormality is deduced when Nc is significantly different from the value expected for the number of genomes present with is deduced from Nt.  
     
     
         10 . Method according to  claim 7 , characterized in tha the so-called control genomic sequence is a sequence present at 2 copies per genome and the presence of a gene abnormality is deduced when Nc and Nt are significantly different.  
     
     
         11 . Method according to one of claims  7 ,  10 , characterized in that the sequence to be assayed corresponds to a trizomie-linked chromosome and in that the control sequence consists of a sequence of another chromosome, the Mc/Nt ratil then being close 1.5 in the case of trisomie.  
     
     
         12 . Method according to  claim 7 , characterized in that the sequence to be assayed corresponds to a deletion of a genome, the control sequence being preferably a sequence of the same chromosome.  
     
     
         13 . Method according to either of claims  1  or  7 , characterized in that the sequence to be assayed is a repeating sequence, the normal sequence then being preferably a sequence of the same chromosome.  
     
     
         14 . Method according to  claim 7 , characterized on that the so-called control genomic sequence and the sequence to be assayed are identical for a given genome, and in that several diferent genomes are combed on the same combing surface and where the respective quantities of genomes of each type are determined.  
     
     
         15 . Method according to  claim 1 , characterized in that: 
 (a) a certain quantity of said genome is attached to and combed on a combing surface,    (b) the combing product is hybridized with probes labeled with radioactive or fluorescent elements and the like, such as beads, particles and the like, corresponding to each clone, such that said probes may be specifically revealed by a color in particular,    (c) the information corresponding to the position of each clone as well as the sizes and the corresponding distances on the genome are extracted,    (d) operations b) and c) are repeated n times by modifying the color, the labeling or the mode of revealing the probes, in the knowledge that with p colors, labelings or different modes of revealing, it is possible to position p clones after n hybridizations.    
     
     
         16 . Method according to one of  claims 1  to  15 , characterized in that probes labeled with modified nucleotides which are optionally revealed separately are used.  
     
     
         17 . Method according to one of  claims 1  to  16 , characterized in that probes labeled by incorporation of nucleotides modified by biotinylation, with DIG or other haptens which are revealed by a system of layers of antibodies or of specific molecules are used.  
     
     
         18 . Method according to one of  claims 1  to  16 , characterized in that probes labeled with fluorescent nucleotides are also used.  
     
     
         19 . Method according to one of  claims 1  to  16 , characterized in that nucleotide probes in which some atoms have been replaced are used.  
     
     
         20 . Method according one of  claims 1  to  19 , characterized in that at least about 10 copies of the genome are placed on the combed surface.  
     
     
         21 . Method according to one of  claims 1  to  14 , characterized in that a sufficient number of signals is present on the combed surface in order to have a statistical error of less than 20% on the Nc/Nt ratio.  
     
     
         22 . Method according one of  claims 1  to  21 , characterized in that the surface contains a calibrating DNA which makes it possible to calibrate each measurement.  
     
     
         23 . Method according one of  claims 2  to  22 , characterized in that the genome is obtained from a sample of biological fluid or from a tissue of biological origin.  
     
     
         24 . Method according one of  claims 2  to  22 , characterized in that the genome is obtained from a biological material containing at least 80% of genetic material of fetal origin.  
     
     
         25 . Kit for diagnosis or for locating genes for carrying out the method according to one of  claims 1  to  24 , characterized in that it contains at least one of the following components: 
 a combing surface,  
 probes which are labeled or which are intended to be labeled, corresponding to the abnormalities to be detected,  
 a device allowing the combing of the DNA,  
 a control genome and/or corresponding control probes, said genome being optionally attached to the surface to be combed,  
 one or more specific results obtained using the preceding protocols in one or more control situations, so as to provide a grid for the interpretation of the results obtained in the diagnoses carried out, for example in the form of an expert system (software for example)  
 an expert system which makes it possible to facilitate the carrying out of diagnoses according to the method of the invention.  
 
     
     
         26 . Diagnostic kit for carrying out the method according to one of  claims 1  to  24 , characterized in that it contains reference standard probes and genetic markers allowing the location of a sequence.  
     
     
         27 . Genomic DNA or genomic DNA portion capable of reacting, under molecular combing conditions, with a probe corresponding to a product of transcription or of translation or of regulation.  
     
     
         28 . Method according to  claim 1 , characterized in that at least one of the probes is a product which is capable of interacting with DNA.  
     
     
         29 . Method according to  claim 1 , characterized in that at least one of the probes is a product of therapeutic interest which is capable of interacting with DNA.  
     
     
         30 . Method according to  claim 1 , characterized in that the reaction of the probe with the combed DNA is modulated by one or more molecules, solvents or other relevant parameters.  
     
     
         31 . Molecule capable of reacting with the genomic DNA capable of being combed, which is isolated by one of the methods previously claimed.  
     
     
         32 . Molecule, solvent or method linked to a parameter identified by one of the methods previously claimed.