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Title: Nucleic acid analysis using terminal-phosphate-labeled nucleotides

Abstract

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog beingmore » added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.« less

Inventors:
 [1];  [1];  [1];  [1];  [1];  [1]
  1. Ithaca, NY
Issue Date:
Research Org.:
Cornell Univ., Ithaca, NY (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
985681
Patent Number(s):
7361466
Application Number:
11/089,871
Assignee:
Cornell Research Foundation, Inc. (Ithaca, NY)
Patent Classifications (CPCs):
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
Y - NEW / CROSS SECTIONAL TECHNOLOGIES Y10 - TECHNICAL SUBJECTS COVERED BY FORMER USPC Y10S - TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
DOE Contract Number:  
066898-0003891
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Korlach, Jonas, Webb, Watt W, Levene, Michael, Turner, Stephen, Craighead, Harold G, and Foquet, Mathieu. Nucleic acid analysis using terminal-phosphate-labeled nucleotides. United States: N. p., 2008. Web.
Korlach, Jonas, Webb, Watt W, Levene, Michael, Turner, Stephen, Craighead, Harold G, & Foquet, Mathieu. Nucleic acid analysis using terminal-phosphate-labeled nucleotides. United States.
Korlach, Jonas, Webb, Watt W, Levene, Michael, Turner, Stephen, Craighead, Harold G, and Foquet, Mathieu. Tue . "Nucleic acid analysis using terminal-phosphate-labeled nucleotides". United States. https://www.osti.gov/servlets/purl/985681.
@article{osti_985681,
title = {Nucleic acid analysis using terminal-phosphate-labeled nucleotides},
author = {Korlach, Jonas and Webb, Watt W and Levene, Michael and Turner, Stephen and Craighead, Harold G and Foquet, Mathieu},
abstractNote = {The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2008},
month = {4}
}

Works referenced in this record:

Near-Field Fluorescence Microscopy Based on Two-Photon Excitation with Metal Tips
journal, May 1999


Reconstruction of DNA sequencing by hybridization
journal, January 2003


Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes
journal, January 1991


Fluorescence Spectroscopy of Single Biomolecules
journal, March 1999


Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution
journal, April 1997


Sensitized Photomodification of Single-Stranded DNA by a Binary System of Oligonucleotide Conjugates
journal, August 1997


Fluorescence of Intramolecular and Intermolecular Interactions of Aminonaphthyl-sulfonate with Nucleotides
journal, August 1982


Theory of Nanometric Optical Tweezers
journal, July 1997


Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay
journal, January 1993


Single-molecule detection as an approach to rapid DNA sequencing
journal, January 1992


Rapid DNA sequencing based upon single molecule detection
journal, February 1991


DNA sequencing with chain-terminating inhibitors
journal, December 1977


Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy
journal, February 1998


Real-Time DNA Sequencing Using Detection of Pyrophosphate Release
journal, November 1996


Feasibility of molecular-resolution fluorescence near-field microscopy using multi-photon absorption and field enhancement near a sharp tip
journal, February 1999


Application of Single Molecule Detection to Dna Sequencing
journal, May 1997