Sequence determination of nucleic acids using electronic detection

• US 7,935,481 B1
• Filed: 07/26/2000
• Issued: 05/03/2011
• Est. Priority Date: 07/26/1999
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Osmetech Technology Inc. (Roche Holding AG)
• Inventors: Terbrueggen, Robert H., Vielmetter, Jost G., Umek, Robert M., Blackburn, Gary, Yu, Changjun, Irvine, Bruce D.
• Primary Examiner(s): Calamita; Heather
• Application Number:
  US09/626,096
• Time in Patent Office: 3,933 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 439/24.3, 439/24.33, 536/231, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention is directed to methods and compositions for the use of self-assembled monolayers to electronically detect nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms (SNPs).

Claims

1. A method of determining the identification of nucleotide(s) at a first detection position in a first domain of a target sequence, said target sequence comprising said first domain and a second domain, said method comprising:

a. providing an electrode with a covalently attached capture probe, wherein said capture probe has a sequence substantially complementary to said second domain of said target sequence;

b. contacting said electrode with;

(i) said target sequence;

(ii) a first label probe substantially complementary to said first domain, comprising a first nucleotide at an interrogation position and a first electron transfer moiety (ETM) with a first redox potential;

(iii) a second label probe substantially complementary to said first domain, comprising a second nucleotide at said interrogation position and a second ETM with a second redox potential;

under conditions wherein if said nucleotide at said interrogation position is perfectly complementary to said detection position, hybridization of said probe(s) occurs; and

c. detecting the presence of said first and/or second ETM to determine the nucleotide(s) at said detection position.

2. The method of claim 1 wherein said method further comprises contacting said electrode with a third label probe substantially complementary to said first domain, comprising a third nucleotide at said interrogation position and a third ETM with a third redox potential.

3. The method of claim 2 wherein said method further comprises contacting said electrode with a fourth label probe substantially complementary to said first domain, comprising a fourth nucleotide at said interrogation position and a fourth ETM with a fourth redox potential.

4. The method of claim 1 wherein said electrode comprises an array of capture probes, each substantially complementary to a second domain of a different target sequence.

5. The method of claim 1 wherein said first label probe contains a plurality of first ETMs.

6. The method of claim 1 wherein said second label probe contains a plurality of second ETMs.

7. The method of claim 1 wherein said electron transfer moieties comprise a transition metal complex.

8. The method of claim 7 wherein said transition metal complex comprises a metallocene.

9. The method of claim 8 wherein said metallocene is a ferrocene.

10. The method of claim 8 wherein said metallocene is a ferrocene derivative.