Method for mapping a nucleic acid

• US 6,713,263 B2
• Filed: 09/09/2002
• Issued: 03/30/2004
• Est. Priority Date: 09/15/1988
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Schwartz, David C
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/238,407
• Publication Number:
  US 20030027201A1
• Time in Patent Office: 568 Days
• Field of Search: 435/6, 436/94
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: G01N 15/02 Investigating particle size or size distribution by measuring osmotic pressure G01N7/10; investigating sedimentation of particle suspensions G01N15/04; investigating individual particles G01N15/10
• CPC Class Codes: G01N 15/10 Investigating individual particles
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/0038 Investigating nanoparticles
• CPC Class Codes: G01N 2015/0288 Sorting the particles
• CPC Class Codes: G01N 2015/1024 Counting particles by non-optical means
• CPC Class Codes: G01N 2015/1028 Sorting particles
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2015/1495 Deformation of particles
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for observing and determining the size of individual molecules and for determining the weight distribution of a sample containing molecules of varying size, which involves placing a deformable or nondeformable molecule in a medium, subjecting the molecule to an external force, thereby causing conformational and/or positional changes, and then measuring these changes. Preferred ways to measure conformational and positional changes include: (1) determining the rate at which a deformable molecule returns to a relaxed state after termination of the external force, (2) determining the rate at which a molecule becomes oriented in a new direction when the direction of the perturbing force is changed, (3) determining the rate at which a molecule rotates, (4) measuring the length of a molecule, particularly when it is at least partially stretched, or (5) measuring at least one diameter of a spherical or ellipsoidal molecule. Measurements of relaxation, reorientation, and rotation rates, as well as length and diameter can be made using a light microscope connected to an image processor. Molecule relaxation, reorientation and rotation also can be determined using a microscope combined with a spectroscopic device. The invention is particularly useful for measuring polymer molecules, such as nucleic acids, and can be used to determine the size and map location of restriction digests. Breakage of large polymer molecules mounted on a microscope slide is prevented by condensing the molecules before mounting and unfolding the molecules after they have been placed in a matrix.

Claims

1. A method for determining a property of a DNA molecule, comprising the steps of

labeling the DNA molecule with a fluorescent agent;

applying an electrical force to elongate a single DNA molecule;

applying exciting light to the DNA molecule to excite fluorescence in the fluorescent agent; and

observing the fluorescence from the DNA molecule to determine a property of the molecule.

2. A method as claimed in claim 1 wherein the property determined is sequence of the DNA molecule.

3. A method as claimed in claim 1 wherein the step of optically isolating the DNA molecule includes elongating or stretching out the DNA molecule.

4. A method as claimed in claim 1 wherein the observing is performed by a microscope.

5. A method as claimed in claim 1 wherein the observing is performed by a spectrographic apparatus.

6. A method for determining one or more properties of a DNA molecule comprising the steps of

attaching a light responsive agent to the DNA molecule;

applying an electrical force to elongate the DNA molecule such that the DNA molecule can be analyzed separately from other DNA molecules;

exciting the DNA molecule with light to excite the light responsive element; and

observing the light from the DNA molecule to determine one or more properties of the DNA molecule.

7. A method as claimed in claim 6 wherein the property determined is sequence of the DNA molecule.

8. A method as claimed in claim 6 wherein the step of manipulating the DNA molecule includes elongating or stretching out the DNA molecule.

9. A method as claimed in claim 6 wherein the observing is performed by a microscope.

10. A method as claimed in claim 6 wherein the observing is performed by a spectrographic apparatus.