Quantitative DNA fiber mapping

• US 5,851,769 A
• Filed: 09/27/1995
• Issued: 12/22/1998
• Est. Priority Date: 09/27/1995
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Weier, Heinz-Ulrich G., Gray, Joe W.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/534,479
• Time in Patent Office: 1,182 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

Claims

1. A method of treating a nucleic acid fiber immobilized to a solid support comprising:

a) providing;

i) a stretched nucleic acid fiber having a sequence immobilized to a solid support; and

ii) one or more labeled probes containing sequences essentially complementary to said sequence of said immobilized nucleic acid fiber;

b) reacting said immobilized nucleic acid fiber with said probes under conditions wherein said probes are capable of binding to essentially complementary sequences present on said nucleic acid fiber and wherein no substantial background is produced; and

c) detecting the presence of probes bound to said nucleic acid fiber.

2. The method of claim 1 wherein said nucleic acid fiber is double-stranded.

3. The method of claim 1 wherein said nucleic acid fiber is single-stranded.

4. The method of claim 1 wherein said nucleic acid fiber is a deoxyribonucleic acid fiber.

5. The method of claim 1 wherein said nucleic acid fiber is a ribonucleic acid fiber.

6. The method of claim 1 wherein said nucleic acid fiber is uniformly stretched to a dimension of approximately 2.3 kilobases per micrometer.

7. The method of claim 1 wherein said solid support comprises a glass support treated with 3-aminopropyltriethoxysilane.

8. The method of claim 1 wherein said labeled probe comprises a probe containing a reporter molecule selected from the group consisting of biotin and digoxigenin.

9. The method of claim 8 wherein the detection of step c) comprises reacting said probe bound to said nucleic acid fiber with a detection reagent selected from the group consisting of AMCA-avidin, FITC-avidin, and rhodamine-labeled anti-digoxigenin antibodies.

10. The method of claim 1 wherein said labeled probe comprises a probe containing a reporter molecule selected from the group consisting of rhodamine and fluorescein isothiocyanate.

11. A method for the stretching of a nucleic acid molecule, comprising:

a) providing;

i) a first solid support treated with 3-aminopropyltriethoxysilane;

ii) a solution comprising a nucleic acid molecule; and

iii) a second solid support; and

b) placing an aliquot of said solution comprising said nucleic acid molecule onto said first solid support;

c) placing said second solid support on top of said aliquot of said solution to cover said solution; and

d) allowing said solution to dry whereby said nucleic acid molecule is uniformly stretched to a dimension of approximately 2.3 kilobases per micrometer.

12. The method of claim 11 wherein said nucleic acid molecule comprises a linear nucleic acid molecule.

13. The method of claim 11 wherein said target nucleic acid molecule comprises a circular nucleic acid molecule.