| US 7,582,421 B2 | ||
| Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip | ||
| Ronald G. Sosnowski, Coronado, Calif. (US); Michael I. Nerenberg, La Jolla, Calif. (US); David M. Canter, San Diego, Calif. (US); Ray R. Radtkey, San Diego, Calif. (US); Ling Wang, San Diego, Calif. (US); and James P. O'Connell, Solana Beach, Calif. (US) | ||
| Assigned to Nanogen, Inc., San Diego, Calif. (US) | ||
| Filed on Sep. 16, 2002, as Appl. No. 10/245,206. | ||
| Application 10/245206 is a continuation of application No. 09/291129, filed on Apr. 12, 1999, granted, now 6,468,742, filed on Oct. 22, 2002. | ||
| Prior Publication US 2003/0073122 A1, Apr. 17, 2003 | ||
| This patent is subject to a terminal disclaimer. | ||
| Int. Cl. C12Q 1/68 (2006.01); C12P 19/34 (2006.01); C07H 21/02 (2006.01); C07H 21/04 (2006.01); C07H 21/00 (2006.01) | ||
| U.S. Cl. 435—6 [435/91.1; 435/91.2; 536/23.1; 536/24.3; 536/24.33; 536/25.3; 536/25.32] | 8 Claims |
| 1. A method for detecting a single nucleotide polymorphism in a sample nucleic acid using an electronically addressable microchip
having a plurality of test sites, wherein each of the test sites comprises an individually controllable electrode covered
by a permeation layer, the method comprising:
providing a sample nucleic acid suspected of containing a single nucleotide polymorphism;
electronically biasing the sample nucleic acid to a test site of the plurality of test sites on the microchip, and concentrating
the sample nucleic acid at the test site;
immobilizing the sample nucleic acid onto the test site; electronically hybridizing a mixture comprising a first probe with
a label selected from the group consisting of fluorescent label, colorimetric label, and chemiluminescent label, and a second
probe with a label selected from the group consisting of fluorescent label, colorimetric label, and chemiluminescent label
to the sample nucleic acid , wherein the first probe is perfectly complementary to the sample nucleic acid and contains a
nucleotide perfectly complementary to the nucleotide at the site of the polymorphism, and forms a first hybridized complex,
and the second probe is complementary to the sample nucleic acid and contains a nucleotide which forms a mismatch with the
nucleotide at the site of the polymorphism, and forms a second hybridized complex, wherein the label of the first probe and
the label of the second probe are different;
subjecting the first and second hybridized complexes to destabilizing conditions sufficient to cause the first probe to dissociate
from the first hybridized complex or the second probe to dissociate from the second hybridized complex if there is at least
one base-pair mismatch between the first probe or the second probe and the sample nucleic acid; and
detecting the first or second hybridized complex following the subjecting step by determining a signal intensity from the
label of the first probe of the first hybridized complex or a signal intensity from the label of the second probe of the second
hybridized complex, wherein the single nucleotide polymorphism in the sample nucleic acid is detected if the signal intensity
from the label of the first probe of the first hybridized complex is present.
|