Nucleic acid sequence detection using multiplexed oligonucleotide PCR

US7153656B2 · kind B2 · utility

Assignee

• LOS ALAMOS NATIONAL SECURITY, LLC · US

Inventors

• John P. Nolan · San Rafael, US
• P. Scott White · Los Alamos, US

Key dates

• Filing date: Jan 3, 2003
• Grant date: Dec 26, 2006
• Expiry date: Oct 24, 2023

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6834
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.