Method for characterizing polymer molecules or the like
First Claim
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.
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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.
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Citations
28 Claims
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1. A method for characterizing single, isolated nucleic acid, including any molecules specifically bound thereto, comprising the steps of:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for determining the molecular weight of single, isolated nucleic acid of unknown size, comprising the steps of:
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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.
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22. A method for characterizing a single, isolated nucleic acid, comprising the steps of:
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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.
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23. A method for mapping a deproteinized, single, isolated nucleic acid or a portion thereof, comprising the steps of:
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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. - View Dependent Claims (24, 25)
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26. A method for sizing and mapping a single, isolated nucleic acid molecule, comprising the steps of:
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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. - View Dependent Claims (27, 28)
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Specification