Cornell Research Foundation Inc.

Mathieu Foquet

Jonas Korlach

Harold G Craighead

Watt W Webb

Michael Levene

Warren Wright

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. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

High speed nucleic acid sequencing

The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

Methods for analyzing nucleic acid sequences

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.

Labeled nucleotide phosphate (NP) probes

Composition for nucleic acid sequencing

Nucleic acid analysis using terminal-phosphate-labeled nucleotides

Method for sequencing nucleic acid molecules

Fabrication techniques for forming arbitrarily shaped fluid capillaries at dimensions below 1 &mgr;m and for forming optical waveguides in the same devices with the same fabrication processes include methods to make integral optical waveguide/fluid-flow systems for greater miniaturization, integration and parallelism of optical excitation and detection systems for the sampling of small volumes. Capillaries with widths below 1 &mgr;m dimensions are fabricated using photolithography and reactive ion etching on glass substrates or by provision of a sacrificial layer, having the configuration desired for a microchannel, covered by a high refractive index material in which a waveguide is formed. Removal of the sacrificial layer produces a microchannel aligned with the waveguide. Dye labeled DNA molecules are driven electrophoretically through the micrometer size channels light emission from and individual molecules can be observed.

Growing community of inventors

Ithaca, NY, United States of America

Mathieu Foquet