Nucleic acid probes, their synthesis and use

• US 9,127,308 B2
• Filed: 02/11/2003
• Issued: 09/08/2015
• Est. Priority Date: 03/07/2002
• Status: Active Grant

• Current Assignee: Binx Health Limited
• Original Assignee: Atlas Genetics Limited
• Inventors: Braven, Helen, Keay, Russell
• Primary Examiner(s): POHNERT, STEVEN C
• Application Number:
  US10/506,958
• Publication Number:
  US 20050221315A1
• Time in Patent Office: 4,592 Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: G01N 2458/30 Electrochemically active labels
• CPC Class Codes: G01N 27/308 at least partially made of carbon
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 27/416 Systems G01N27/27 takes precedence
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The invention provides a method of probing for a nucleic acid comprising: contacting a nucleic acid solution with an oligonucleotide probe labelled with an electrochemically active marker, providing conditions at which the probe is able to at least partially hybridize with any complementary target sequence which may be present in the nucleic acid solution, selectively degrading either hybridized, partially hybridized or unhybridized nucleic acid probe, and electrochemically determining information relating to the electrochemically active marker. The invention further provides novel molecules with use in methods of the invention.

Claims

1. A method of detecting a target nucleic acid sequence in a nucleic acid solution comprising:

contacting a nucleic acid solution with an oligonucleotide probe labeled at the 5′

-terminal with an electrochemically active marker;

hybridizing the oligonucleotide probe with a complementary target sequence present in the nucleic acid solution and thereby forming a double stranded hybridized nucleic acid;

degrading the double stranded hybridized nucleic acid by digestion with a duplex specific 5′

to 3′

exonuclease to form a mononucleotide degraded probe labeled with the electrochemically active marker or a dinucleotide probe labeled with the electrochemically active marker;

electrochemically discriminating a redox based signal of the mononucleotide probe or the dinucleotide probe from any undegraded or unhybridized oligonucleotide probe in the solution, wherein the redox based signal is influenced by at least the size of the probe to which the electrochemically active marker is attached; and

detecting the target nucleic acid based on the discrimination of the redox based signal from the mononucleotide probe or the dinucleotide probe from any undegraded or unhybridized oligonucleotide probe.

2. The method as claimed in claim 1, wherein detecting the target sequence is used for the detection of nucleic acid polymorphisms.

3. The method as claimed in claim 1, wherein detecting the target sequence is used for detection of allelic polymorphisms.

4. The method as claimed in claim 1, wherein detecting the target sequence is used for the detection of single nucleotide polymorphisms.

5. The method as claimed in claim 1, wherein detecting the target sequence is used for the quantification of nucleic acid species.

6. The method as claimed in claim 1, wherein detecting the target sequence is used for the quantification of gene expression.

7. The method as claimed in claim 1, in which two or more oligonucleotide probes each complementary to different target sequences in the nucleic acid solution are used, wherein each probe is labeled with a different electrochemically active marker.

8. The method as claimed in claim 7, in which the two or more electrochemically active markers have peaks in their voltammogram traces that are resolvable from each other.

9. The method as claimed in claim 1, wherein the 5′

to 3′

exonuclease is T7 exonuclease.

10. The method as claimed in claim 1, wherein the step of electrochemically discriminating the redox based signal of the mononucleotide probe, the dinucleotide probe, from any undegraded or unhybridized oligonucleotide probe is by voltammetry.

11. The method as claimed in claim 1, wherein the step of electrochemically discriminating the redox based signal of the mononucleotide probe or the dinucleotide probe from any undegraded or unhybridized oligonucleotide probe is by an amperometric technique.

12. The method as claimed in claim 1, wherein the step of electrochemically discriminating the signal of the mononucleotide probe or the dinucleotide probe, from any undegraded or unhybridized oligonucleotide probe is by differential pulse voltammetry.