Electrochemical detection of mismatch nucleic acids

• US 7,291,457 B2
• Filed: 06/21/2002
• Issued: 11/06/2007
• Est. Priority Date: 06/21/2001
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Wang, Tza-Huei, Ho, Chih-Ming, Huang, Jun, Miller, Jeffrey F., Liu, Minghsun
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US10/177,537
• Publication Number:
  US 20040018492A1
• Time in Patent Office: 1,964 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

A method and system for specifically detecting perfectly matched probe-target hybrids by annealing a hairpin-based probe to a nucleic acid target under conditions favoring the formation of perfectly matched targets over mismatched targets, and electrochemically detecting the annealed probe. Sensitivity of detection can be enhanced by catalyzed reporter deposition of probe signal prior to electrochemical detection. Specificity can be enhanced by including hairpin-based competitor probes in the annealing step. The detection assay is particularly applicable to typing single nucleotide polymorphisms by DNA sandwich hybridization.

Claims

1. A method of specifically detecting perfectly matched probe-target hybrids, comprising the steps of:

annealing a hairpin-based detector probe to a nucleic acid target under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids;

electrochemically detecting annealed probe signal by sensing an electro-reductive or electro-oxidative process at an electrode, said signal generated by horse radish peroxidase-catalyzed reduction of at electrochemically active substrate at one end, and

wherein the detector probe is attached at the other end to benzbydroxamic acid.

2. A method of typing a single nucleotidc polymorphism of a nucleic acid target sequence, comprising the steps of:

annealing each of at least two hairpin-based detector probes in separate reactions to the target sequence under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids, one probe forming a perfectly matched hybrid with the target sequence, each probe comprising a sequence complementary to the target sequence, the complementary sequences of the probes differing from one another at a single nucleotide position;

electrochemically detecting annealed probe signal at one end by sensing an electro-reductive or electro-oxidative process at an electrode, said signal generated by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate, and

wherein each detector probe is attached at the other end to benzhydroxamic acid.