AMPLIFICATION OF NUCLEIC ACIDS WITH ELECTRONIC DETECTION

• US 20020006643A1
• Filed: 01/27/1999
• Published: 01/17/2002
• Est. Priority Date: 01/27/1998
• Status: Active Grant

• Current Assignee: Osmetech Technology Inc. (Roche Holding AG)
• Original Assignee: Clinical Micro Sensors Inc. (Roche Holding AG)
• Inventors: BAMDAD, CYNTHIA, KAYYEM, JON FAIZ
• Granted Patent: US 7,090,804 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: B01D 17/0214 with removal of one of the phases
• CPC Class Codes: B01D 17/045 with coalescers
• CPC Class Codes: B01D 24/105 downward filtration without specifications about the filter material supporting means
• CPC Class Codes: B01D 24/4642 with valves, e.g. rotating valves
• CPC Class Codes: B01D 24/48 integrally combined with devices for controlling the filtration
• CPC Class Codes: B01D 35/12 Devices for taking out of action one or more units of multi- unit filters, e.g. for regeneration
• 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: C02F 1/283 using coal, charred products, or inorganic mixtures containing them
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48

Abstract

The invention relates to compositions and methods useful in the detection of nucleic acids using a variety of amplification techniques, including both signal amplification and target amplification. Detection proceeds through the use of an electron transfer moiety (ETM) that is associated with the nucleic acid, either directly or indirectly, to allow electronic detection of the ETM using an electrode.

Claims

1. A method for detecting a first target nucleic acid sequence comprising:

a) hybridizing at least a first primer nucleic acid to said first target sequence to form a first hybridization complex;

b) contacting said first hybridization complex with a first enzyme to form a modified first primer nucleic acid;

c) disassociating said first hybridization complex;

d) forming a first assay complex comprising at least one ETM and said modified first primer nucleic acid, wherein said first assay complex is covalently attached to an electrode; and

e) detecting electron transfer between said ETM and said electrode as an indication of the presence of said target sequence.

2. A method according to claim 1 wherein steps a) through c) are repeated prior to step d).

3. A method according to claim 1 further comprising:

a) hybridizing at least a second primer nucleic acid to a second target sequence that is substantially complementary to said first target sequence to form a second hybridization complex;

b) contacting said second hybridization complex with said first enzyme to form a modified second primer nucleic acid;

c) disassociating said second hybridization complex; and

d) forming a second assay complex comprising at least one ETM and said modified second primer nucleic acid, wherein said second assay complex is covalently attached to an electrode.

4. A method according to claim 1 wherein steps a) through c) are repeated prior to step d).

5. A method according to claim 2 wherein said first enzyme comprises a DNA polymerase and said modification is an extension of said primer such that the polymerase chain reaction (PCR) occurs.

6. A method according to claim 2 wherein said first enzyme comprises a ligase and said modification comprises a ligation of said first primer which hybridizes to a first domain of said first target sequence to a third primer which hybridizes to a second adjacent domain of said first target sequence, such that the ligase chain reaction (LCR) occurs.

7. A method according to claim 3 wherein said first enzyme comprises a ligase and said modification is a ligation of said second primer which hybridizes to a first domain of said second target sequence to a fourth primer which hybridizes to a second adjacent domain of said second target sequence, such that the ligase chain reaction (LCR) occurs.

8. A method according to claim 2 wherein said first primer comprises a first probe sequence, a first scissile linkage and a second probe sequence, wherein said first enzyme will cleave said first scissile linkage resulting in the separation of said first and said second probe sequences and the disassociation of said first hybridization complex, leaving said first target sequence intact, such that the cycling probe technology (CPT) reaction occurs.

9. A method according to claim 4 wherein said second primer comprises a third probe sequence, a second scissile linkage and a fourth probe sequence, wherein said first enzyme will cleave said second scissile linkage resulting in the separation of said third and said fourth probe sequences and the disassociation of said second hybridization complex, leaving said second target sequence intact, such that the cycling probe technology (CPT) reaction occurs.

10. A method according to claim 2 wherein said first enzyme is a polymerase that extends said first primer and said modified first primer comprises a first newly synthesized strand, and said method further comprises:

a) the addition of a second enzyme comprising a nicking enzyme that nicks said extended first primer leaving said first target sequence intact; and

b) extending from said nick using said polymerase, thereby displacing said first newly synthesized strand and generating a second newly synthesized strand;

such that strand displacement amplification (SDA) occurs.

11. A method according to claim 4 wherein said first enzyme is a polymerase that extends said second primer and said modified first primer comprises a third newly synthesized strand, and said method further comprises:

a) the addition of a second enzyme comprising a nicking enzyme that nicks said extended second primer leaving said second target sequence intact; and

b) extending from said nick using said polymerase, thereby displacing said third newly synthesized strand and generating a fourth newly synthesized strand;

such that strand displacement amplification (SDA) occurs.

12. A method according to claim 2 wherein said first target sequence is a RNA target sequence, said first primer nucleic acid is a DNA primer comprising an RNA polymerase promoter, said first enzyme is a reverse-transcriptase that extends said first primer to form a first newly synthesized DNA strand, and said method further comprises:

a) the addition of a second enzyme comprising an RNA degrading enzyme that degrades said first target sequence;

b) the addition of a third primer that hybridizes to said first newly synthesized DNA strand;

c) the addition of a third enzyme comprising a DNA polymerase that extends said third primer to form a second newly synthesized DNA strand, to form a newly synthesized DNA hybrid;

d) the addition of a fourth enzyme comprising an RNA polymerase that recognizes said RNA polymerase promoter and generates at least one newly synthesized RNA strand from said DNA hybrid;

such that nucleic acid sequence-based amplification (NASBA) occurs.

13. A method for detecting a target nucleic acid sequence comprising:

a) forming a first hybridization complex comprising an amplifier probe and a target sequence, wherein said amplifier probe comprises at least two amplification sequences;

b) hybridizing a first portion of at least one label probe to all or part of at least one amplification sequence;

c) hybridizing a second portion of said label probe to a detection probe covalently attached to an electrode via a conductive oligomer to form a second hybridization complex that contains at least a first electron transfer moiety (ETM); and

d) detecting said label probe by measuring electron transfer between said first ETM and said electrode.

14. A method for detecting a target nucleic acid sequence comprising:

a) forming a first hybridization complex comprising an amplifier probe and a target sequence, wherein said amplifier probe comprises at least two amplification sequences, wherein said first hybridization complex is covalently attached to an electrode comprising a monolayer comprising conductive oligomers;

b) hybridizing at least one label probe comprising at least one electron transfer moiety (ETM) to all or part of at least one amplification sequence;

c) detecting said label probe by measuring electron transfer between said first ETM and said electrode.

15. A kit for the detection of a first target nucleic acid sequence comprising:

a) at least a first nucleic acid primer substantially complementary to at least a first domain of said target sequence;

b) at least a first enzyme that will modify said first nucleic acid primer; and

c) an electrode comprising at least one detection probe covalently attached to said electrode via a conductive oligomer.

17. A kit according to claim 15 or 16 for the detection of a PCR reaction wherein said first enzyme is a thermostabile DNA polymerase.

18. A kit according to claim 15 or 16 for the detection of a LCR reaction wherein said first enzyme is a ligase and said kit comprises a first nucleic acid primer substantially complementary to a first domain of said first target sequence and a third nucleic acid primer substantially complementary to a second adjacent domain of said first target sequence.

19. A kit according to claim 15 or 16 for the detection of a strand displacement amplification (SDA) reaction wherein said first enzyme is a polymerase and said kit further comprises a nicking enzyme.

20. A kit according to claim 15 or 16 for the detection of a NASBA reaction wherein said first enzyme is a reverse transcriptase, and said kit comprises a second enzyme comprising an RNA degrading enzyme, a third primer, a third enzyme comprising a DNA polymerase and a fourth enzyme comprising an RNA polymerase.

16. A kit for the detection of a first target nucleic acid sequence comprising:

a) at least a first nucleic acid primer substantially complementary to at least a first domain of said target sequence;

b) at least a first enzyme that will modify said first nucleic acid primer; and

c) an electrode comprising a monolayer comprising conductive oligomers.