Image processing and analysis of individual nucleic acid molecules

• US 5,720,928 A
• Filed: 04/03/1995
• Issued: 02/24/1998
• Est. Priority Date: 09/15/1988
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: New York University
• Inventors: Schwartz, David C.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/415,710
• Time in Patent Office: 1,058 Days
• Field of Search: 435/6, 536/23.1, 422/55, 422/58, 422/99, 422/129, 422/186
• US Class Current: 422/186
• 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 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• 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 2015/1497 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

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 system for characterizing a nucleic acid molecule, comprising:

(a) the nucleic acid molecule elongated and fixed onto a planar surface so that the nucleic acid molecule remains accessible for enzymatic reactions and/or hybridization reactions;

(b) an enzyme fixed onto the planar surface;

(c) a divalent cation cofactor included on the planar surface, wherein the cofactor is required for activity of the fixed enzyme and is reversibly chelated with a divalent cation chelator to protect the fixed enzyme from being activated; and

(b) a device for imaging the elongated and fixed nucleic acid molecule to obtain its physical characteristics.

2. The system of claim 1 in which the enzyme is a restriction endonuclease, an exonuclease, a polymerase, a ligase or a helicase.

3. The system of claim 1 in which the cofactor is released upon exposure to a specific wavelength of light, and the fixed enzyme is activated in the location of the exposure.