Back to results
US 6,607,888 B2Granted
Method for analyzing nucleic acid reactions
AssigneeWisconsin Alumni Research Foundation
InventorsDavid C. Schwartz, Tian Wu
Life of this patent
Estimating the term…
Abstract
Disclosed are methods for analyzing reactions involving nucleic acids. The invention utilizes nucleic acids immobilized on a defined substrate in such a manner that the nucleic acid can participate in enzymatic and chemical reactions. These reactions are carried out in the presence of labeled reagents, thereby enabling the progress of the reactions to be analyzed using various techniques, such a fluorescent microscopy. The invention is particularly well suited for investigating transcription phenomena and generating genome-wide maps based upon reactions of individual nucleic acid molecules.
Claims42
A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:
- (a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid molecules in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;
- then
- (b) subjecting the elongated and fixed nucleic acid of step (a) to an enzymatic or chemical reaction in the presence of a labeled reagent that generates signals correlating to the enzymatic or chemical reaction;
- and then
- (c) acquiring and compiling the signals generated by the labeled reagent, whereby the enzymatic or chemical reaction of step (b) is analyzed.
2 more elements
A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:
- (a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid molecules in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;
- then
- (b) subjecting the elongated and fixed nucleic acids of step (a) to an enzymatic transcription reaction in the presence of a labeled reagent that generates signals correlating to the enzymatic transcription reaction;
- and then
- (c) acquiring and compiling the signals generated by the labeled reagent, whereby the enzymatic transcription reaction of step (b) is analyzed.
2 more elements
A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:
- (a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid molecules in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;
- then
- (b) subjecting the elongated and fixed nucleic acid of step (a) to a transcription reaction followed by a restriction reaction in the presence of a labeled reagent that generates signals correlating to the transcription reaction and the restriction reaction, respectively;
- then
- (c) acquiring and compiling the signals generated by the labeled reagent;
- then
- (d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, then:
- (i) observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule;
- and
- (ii) observing the individual elongated nucleic acid molecule of step (d) (i) for appearance of gaps corresponding to cleavage sites between restriction fragments;
- then
- (e) reiteratively repeating steps (d), (d) (i), and (d) (ii) on additional individual elongated nucleic acid molecules, to thereby generate additional images, and then
- (f) compiling an ordered map correlating transcription event sites and restriction enzyme cleavage sites based upon the images.
10 more elements
A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:
- (a) subjecting nucleic acid molecules to an enzymatic or chemical reaction in the presence of a labeled reagent that generates signals correlating to the enzymatic or chemical reaction, thereby generating nucleic acid reaction products;
- and then
- (b) elongating and fixing onto a surface of a substrate a plurality of the nucleic acid reaction products of step (a) in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for further enzymatic and chemical reactions;
- then
- (c) acquiring and compiling the signals generated by the labeled reagent, whereby the enzymatic or chemical reaction of step (a) is analyzed.
2 more elements
A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:
- (a) subjecting a nucleic acid to a transcription reaction followed by a restriction reaction in the presence of a labeled reagent that generates signals correlating to the transcription and the restriction reaction, respectively, thereby generating nucleic acid reaction products;
- then
- (a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid reaction products of step (a) in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;
- then
- (c) acquiring and compiling the signals generated by the labeled reagent;
- then
- (d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, then:
- (i) observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule;
- and
- (ii) observing the individual elongated nucleic acid molecule of step (d) (i) for appearance of gaps corresponding to cleavage sites between restriction fragments;
- then
- (e) reiteratively repeating steps (d), (d) (i), and (d) (ii) on additional individual elongated nucleic acid molecules, to thereby generate additional images, and then
- (f) compiling an ordered map correlating transcription event sites and restriction enzyme cleavage sites based upon the images.
10 more elements
Dependencies are inferred from the claim text.