Physical mapping method using molecular combing technique allowing positioning of a great number of clones within a genome

• US 7,368,234 B2
• Filed: 03/11/2003
• Issued: 05/06/2008
• Est. Priority Date: 10/30/1996
• Status: Expired due to Fees

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Bensimon, Aaron, Michalet, Xavier, Bensimon, David
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/386,354
• Publication Number:
  US 20040033510A1
• Time in Patent Office: 1,883 Days
• Field of Search: 435/6, 436/94
• 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

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 where the method can include: (a) fixing and combing a certain amount of the B DNA on a combing surface; (b) reacting the product of step (a) 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 hybridized probes) and determining from this extracted information the presence, the location and/or the amount of a gene(s) or specific A DNA sequences. This method can be used in particular for the diagnosis of genetic diseases.

Claims

1. A method of determining the position of pⁿ cloned nucleotide sequences within a genome, comprising:

(a) providing a surface on which genomic DNA has been aligned using a molecular combing technique with the aid of a receding meniscus;

(b) contacting the aligned genomic DNA with labeled probes, wherein the labeled probes comprise nucleotide sequences that are complementary to the cloned nucleotide sequences;

(c) detecting hybridization between the labeled probes and the genomic DNA; and

(d) determining the position of the labeled probes, the distance between the labeled probes, or the size of the labeled probes, and deducing therefrom the position of the pⁿ cloned nucleotide sequences,

wherein parts b) and c) are repeated “

n”

times by modifying the color, the labeling, or the mode of revealing the probes, so that if the number of colors, labelings, or modes of revealing equals “

p”

, it is possible to position pⁿ cloned nucleotide sequences after “

n”

hybridizations,

wherein the position of the labeled probes, the distance between the labeled probes, or the size of the labeled probes is converted into a histogram.

2. The method according to claim 1, wherein the label is fluorescent or radioactive.

3. The method according to claim 1, wherein the label is fluorescent.

4. The method of claim 1, wherein the probes are labeled with modified nucleotides.

5. The method according to claim 4, wherein the modified nucleotides are modified with biotin, digoxigenin (DIG), or other haptens.

6. The method of claim 1, wherein the probes are nucleotide probes in which some atoms have been replaced.

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

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

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

10. The method of claim 9, wherein the genomic DNA is obtained from a biological material containing at least 80% of genetic material of fetal origin.