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

• US 7,732,143 B2
• Filed: 03/18/2008
• Issued: 06/08/2010
• Est. Priority Date: 10/30/1996
• Status: Expired due to Fees

• Current Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Original Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Inventors: Michalet, Xavier, Bensimon, David, Bensimon, Aaron
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US12/076,408
• Publication Number:
  US 20090123926A1
• Time in Patent Office: 812 Days
• Field of Search: 435/6
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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 characterised 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 sizes for quantifying the number of hybridised probes) for determining therefrom 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.

Claims

1. A method of identifying a genetic abnormality comprising a break in a genome, wherein the method comprises:

(a) providing a surface on which genomic DNA comprising a plurality of clones has been aligned using a molecular combing technique;

(b) contacting the genomic DNA with at least one probe that is specific for a genomic sequence for which the genetic abnormality is sought;

(c) detecting a hybridization signal between the at least one probe and the genomic DNA;

(d) identifying the presence of the break in the genome directly or by comparing the length of the sequences detected by the hybridization signal to the length of sequences detected by a hybridization signal obtained using a control genome that does not contain the break and the at least one probe of part (b), and

(e) determining the number of clones having a defined probe length, wherein the determined numbers of clones and the lengths of the sequences detected by the hybridization signals are converted into a graph.

2. The method of claim 1, wherein the at least one probe is labeled.

3. The method of claim 1, wherein the at least one probe overlaps a portion of the genome not involved in the genetic abnormality.

4. The method of claim 1, wherein the histogram is compared with a second histogram that corresponds to the length of hybridized sequences obtained using the control genome and the at least one probe of part (b).

5. The method of claim 1, wherein the break in the genome comprises a repeating sequence, a deleted sequence, a translocated sequence, or an inserted sequence.

6. The method of claim 1, wherein the at least one probe is labeled with a modified nucleotide.

7. The method according to claim 6, wherein the modified nucleotide is modified with biotin, digoxigenin (DIG), or other haptens.

8. The method of claim 1, wherein the at least one probe is labeled with a fluorescent label.

9. The method of claim 1, wherein the at least one probe is a nucleotide probe in which some atoms have been replaced.

10. The method of claim 1, wherein the surface comprises at least about 10 copies of the genomic DNA.

11. The method of claim 1, wherein the surface further comprises a calibrating DNA, for calibrating a measurement.

12. The method of claim 1, wherein the genomic DNA is obtained from a sample of biological fluid or from a tissue of biological origin.

13. The method of claim 12, wherein the genomic DNA is obtained from a biological material containing at least 80% of genetic material of fetal origin.

14. The method of claim 1, wherein the graph is a histogram.