Electrochemical detection of single base extension

• US 6,518,024 B2
• Filed: 12/13/1999
• Issued: 02/11/2003
• Est. Priority Date: 12/13/1999
• Status: Expired due to Term

• Current Assignee: Osmetech Technology Inc. (Roche Holding AG)
• Original Assignee: Motorola, Inc. (Motorola Solutions, Inc.)
• Inventors: Gallagher, Sean, Shi, Song, Choong, Vi-En, Maracas, George
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): KIM, YOUNG J
• Application Number:
  US09/459,685
• Publication Number:
  US 20020064775A1
• Time in Patent Office: 1,156 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

This invention relates to apparatus and methods for detecting single base extension to an oligonucleotide array using electrochemical labels.

Claims

1. A method for identifying a base at an interrogation position in a target sequence of a sample, said method comprising:

a) contacting said sample with an oligonucleotide array comprising a plurality of microelectrodes each comprising a polyacrylamide gel pad and a different immobilized oligonucleotide probe, under conditions whereby said target sequence hybridizes to at least one of said oligonucleotide probes at a position adjacent to said interrogation position to form a hybridization complex;

b) contacting said hybridization complex with a hybridization solution comprising;

i) a polymerase; and

ii) different primer extension units each comprising a chain-terminating nucleotide and a different electrochemical reporter group;

under conditions whereby a primer extension unit is added to the terminus of said probe in said hybridization complex; and

c) detecting the presence of said electrochemical reporter group to identify the base at said interrogation position.

2. A method according to claim 1 wherein at least one of said electrochemical reporter groups is ferrocene.

3. A method according to claim 1 wherein at least one of said electrochemical reporter groups comprises transition metal complex.

4. A method according to claim 3 wherein said transition metal complex comprises a transition metal ion selected from the group consisting of ruthenium, iron, cobalt and osmium.

5. A method according to claim 1 wherein said chain-terminating nucleotides are dideoxyribonucleotides.

6. A method according to claim 1 wherein said chain-terminating nucleotides are acyclonucleotides.

7. A method according to claim 1 wherein said electrochemical reporter groups are covalently linked to said chain-terminating nucleotides using a linker moiety.

8. A kit used for identifying a base at an interrogation position in a target sequence of a sample, said kit comprising:

a) an apparatus comprising;

i) an oligonucleotide array comprising a plurality of microelectrodes each comprising a polyacrylamide gel pad and a different immobilized oligonucleotide probe,

ii) a voltage source and

iii) a detector to detect the presence of an electrochemical reporter label;

b) a hybridizing solution comprising;

i) chain-terminating nucleotides labeled with different electrochemical reporters, and

ii) a polymerase.

9. A kit according to claim 8 wherein at least one of said electrochemincal reporter groups is ferrocene.

10. A kit according to claim 8 wherein at least one of said electrochemical reporter groups comprises a transition metal complex.

11. A kit according to claim 10 wherein said transition metal complex comprises a transition metal ion selected from the group consisting of ruthenium, iron, cobalt and osmium.

12. A kit according to claim 8 wherein said chain-terminating nucleotides are dideoxyribonucleotides.

13. A kit according to claim 8 wherein said chain-terminating nucleotides are acyclonucleotides.

14. A kit according to claim 8 wherein said electrochemical reporter groups are covalently linked to said chain-terminating nucleotides using a linker moiety.