Method for characterizing polymer molecules or the like

• US 5,599,664 A
• Filed: 12/07/1993
• Issued: 02/04/1997
• Est. Priority Date: 04/05/1989
• 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): Zitomer, Stephanie W.
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/162,379
• Time in Patent Office: 1,155 Days
• Field of Search: 435/6, 435/172.3, 435/270, 435/91.1, 435/183, 435/820, 435/968, 204/182.8, 935/1, 935/19, 935/76, 935/77, 436/543-546, 436/800, 436/94, 536/23.1, 536/24.3-24.32, 536/25.3
• US Class Current: 435/6.16
• 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 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• 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 27/44721 by optical means
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: Y10S 435/82 Subcellular parts of microorganisms
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 977/853 Biological sample
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for observing and determining the size of individual particles and for determining the weight distribution of a sample containing particles of varying size, which involves placing a deformable or nondeformable particle in a medium, subjecting the particle 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 particle returns to a relaxed state after termination of the external force, (2) determining the rate at which a particle becomes oriented in a new direction when the direction of the perturbing force is changed, (3) determining the rate at which a particle rotates, (4) measuring the length of a particle, particularly when it is at least partially stretched, or (5) measuring at least one diameter of a spherical or ellipsoidal particle. 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. Particle 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 characterizing single, isolated nucleic acid, including any molecules specifically bound thereto, comprising the steps of:

placing said nucleic acid in a medium,

applying a temporary external force in a repeatable, controlled fashion to said nucleic acid, said force being of a type which causes a molecular size-dependent measurable change in said nucleic acid, wherein said change comprises a conformational change or a change in position,

observing said nucleic acid using a microscope;

measuring said change using the microscope; and

determining the molecular size of said nucleic acid based on said change measured using the microscope.

2. The method of claim 1 wherein said change comprises a conformational change, and the rate of change after termination of said force is measured.

3. The method of claim 1 wherein said change comprises a conformational change, and the rate of change after initiation of said force is measured.

4. The method of claim 1 wherein said change comprises a positional change, and the rate of change after initiation of said force is measured.

5. The method of claim 4 wherein said positional change comprises a rotational change and optionally comprises lateral movement, and a rate of said positional change after initiation of said force is measured.

6. The method of claim 1 further comprising the step of collapsing said nucleic acid before placement in said medium.

7. The method of claim 6, wherein said nucleic acid is shear sensitive.

8. The method of claim 6, further comprising the step of uncollapsing said nucleic acid after placement in said medium.

9. The method of claim 8 wherein said nucleic acid is collapsed using a condensation agent, and is uncollapsed using an ionic substance.

10. The method of claim 9, wherein said condensation agent comprises spermine, spermidine, alcohol or hexamine cobalt.

11. The method of claim 1, wherein said medium comprises at least one of a solution, gel, powder and glass.

12. The method of claim 1, wherein said nucleic acid is labelled with a labelling agent before it is measured.

13. The method of claim 12, wherein said labelling agent comprises a stain.

14. The method of claim 1 wherein said microscope is a light microscope.

15. The method of claim 1, wherein said medium is a matrix.

16. The method of claim 15 further comprising the step of collapsing said molecule before placement in said matrix.

17. The method of claim 16, wherein said molecule is shear sensitive.

18. The method of claim 17 further comprising the step of uncollapsing said molecule after placement in said matrix.

19. The method of claim 18 wherein said molecule is collapsed using a condensation agent, and is uncollapsed using an ionic substance.

20. The method of claim 1, wherein said measuring and determining steps further comprise using the microscope linked to a computerized image processor.

21. A method for determining the molecular weight of single, isolated nucleic acid of unknown size, comprising the steps of:

placing a first nucleic acid of predetermined size in medium;

applying a temporary external force in a repeatable, controlled fashion to said first nucleic acid of predetermined size, said force being of a type which causes a molecular size-dependent measurable change in said first nucleic acid of predetermined size, wherein said change comprises a conformational change or a change in position;

observing said first nucleic acid of predetermined size using a microscope;

measuring said change using the microscope;

placing a second nucleic acid of predetermined size in a medium;

applying a temporary external force in a repeatable, controlled fashion to said second nucleic acid of predetermined size, said force being of a type which cause a molecular size-dependent measurable change in said second nucleic acid of predetermined size, wherein said change comprises a conformational change or a change in position;

observing said second nucleic acid of predetermined size using the microscope;

measuring said change using the microscope;

placing a nucleic acid of unknown size in a medium;

applying a temporary external force in a repeatable;

controlled fashion to said nucleic acid of unknown size, said force being of a type which causes a molecular size-dependent measurable change in said nucleic acid of unknown size, wherein said change comprises a conformational change or a change in position;

observing said nucleic acid of unknown size using the microscope;

measuring said change using the microscope;

calculating a mathematical relationship between the size and rate of change of each of said nucleic acids of predetermined size; and

determining the size of said nucleic acid of unknown size.

22. A method for characterizing a single, isolated nucleic acid, comprising the steps of:

placing said nucleic acid in a medium;

placing in said medium a recombinational enzyme and a labelled probe that hybridizes to a portion of said nucleic acid, wherein said recombinational enzyme facilitates the hybridization of the labelled probe to a portion of said nucleic acid;

hybridizing said probe to said nucleic acid, whereby a complex is formed;

observing said nucleic acid using a microscope; and

mapping said complex using the microscope.

23. A method for mapping a deproteinized, single, isolated nucleic acid or a portion thereof, comprising the steps of:

placing in a medium said nucleic acid and a probe to a portion of said nucleic acid;

inducing hybridization;

observing said nucleic acid using a microscope; and

characterizing said hybridized nucleic acid using the microscope.

24. The method of claim 23, further comprising contacting said nucleic acid with a recombinational enzyme to facilitate hybridization of said probe to a portion of said nucleic acid under conditions such that said hybridization occurs.

25. The method of claim 24, wherein said probe is labelled.

26. A method for sizing and mapping a single, isolated nucleic acid molecule, comprising the steps of:

i) placing said molecule in a medium;

ii) contacting said molecule with at least one restriction enzyme under conditions such that restriction digestion occurs, whereby fragments are produced;

iii) observing said digestion using a microscope;

iv) applying a temporary external force to said fragments, said force being of a type that causes a molecular size-dependent measurable change in said fragments;

v) measuring said change using the microscope; and

vi) determining the molecular size of said fragments based on said change measured using the microscope.

27. The method according to claim 26 wherein said nucleic acid is mounted on a microscope slide and stretched prior to said restriction digestion.

28. The method of claim 26 wherein said change is a conformational change.