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Title: Method for sequencing nucleic acid molecules

Patent ·
OSTI ID:1175774

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.

Research Organization:
Cornell Univ., Ithaca, NY (United States)
Sponsoring Organization:
USDOE
Assignee:
Cornell Research Foundation, Inc. (Ithaca, NY)
Patent Number(s):
7,056,661
Application Number:
09/572,530
OSTI ID:
1175774
Country of Publication:
United States
Language:
English

References (13)

Near-Field Fluorescence Microscopy Based on Two-Photon Excitation with Metal Tips journal May 1999
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
DNA SEQUENCING:A Sequencing Method Based on Real-Time Pyrophosphate journal July 1998
Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution journal April 1997
Techniques for single molecule sequencing journal September 1997
Sensitized Photomodification of Single-Stranded DNA by a Binary System of Oligonucleotide Conjugates journal August 1997
Theory of Nanometric Optical Tweezers journal July 1997
Single-molecule detection as an approach to rapid DNA sequencing journal January 1992
DNA sequencing with chain-terminating inhibitors journal December 1977
PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing journal July 1997
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

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