Nucleic Acid Probes, their Synthesis and Use
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 hybridise with any complementary target sequence which may be present in the nucleic acid solution, selectively degrading either hybridised, partially hybridised or unhybridised 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.
1 . A method of electrochemically detecting a nucleic acid in a sample with an electrode, the method comprising:
contacting a nucleic acid sample in a container comprising said electrode with an oligonucleotide probe comprising an oligonucleotide and an electrochemically active marker and hybridizing the oligonucleotide probe with any complementary target sequence present in the nucleic acid sample;
digesting the hybridized, nucleic acid in the sample with a duplex-specific nuclease to form a mononucleotide and/or a dinucleotide labeled with the electrochemically active marker; wherein the mononucleotide and/or the dinucleotide migrate to the electrode in the container, and wherein the
electrochemical signal of the electrochemically active marker on the mononucleotide and/or dinucleotide is detected at the electrode.
2 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used to derive information concerning the presence or absence of at least one nucleic acid species.
3 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used to quantify relative proportions of digested and non-digested probe.
4 . (canceled)
5 . The method as claimed in claim 1 , wherein the duplex-specific nuclease is an endonuclease.
6 . (canceled)
7 . The method as claimed in claim 1 , wherein the duplex-specific nuclease is a deoxyribonuclease.
8 . (canceled)
9 . The method as claimed in claim 1 , wherein the duplex-specific nuclease is an exonulcease.
10 . The method as claimed in claim 9 , wherein the duplex-specific nuclease is T7 exonuclease.
11 . (canceled)
12 . The method as claimed in claim 1 wherein the duplex-specific nuclease is a 5′ nuclease.
13 . The method as claimed in claim 12 , wherein the 5′ nuclease is a DNA polymerase.
14 . The method as claimed in claim 13 , wherein the DNA polymerase is a thermostable enzyme.
15 . The method as claimed in claim 14 , wherein the thermostable enzyme is Taq polymerase.
16 . The method as claimed in claim 14 , wherein the nucleic acid sample also comprises a pair of primers suitable for extension by the DNA polymerase.
17 . The method as claimed in claim 16 , wherein reaction conditions and temperature cycling are suitable for a polymerase chain reaction (PCR) to take place concomitant to the 5′ nuclease digestion of probe.
18 .- 19 . (canceled)
20 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker detects nucleic acid polymorphisms.
21 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker detects allelic polymorphisms.
22 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker detects single nucleotide polymorphisms.
23 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used for the quantification of nucleic acid species.
24 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used for the quantification of gene expression.
25 .- 42 . (canceled)
43 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used for the detection of a genetic disease or a genetic disease carrier status or a genetic predisposition to disease.
44 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used to detect or identify a pathogen in a sample.
45 . The method as claimed in claim 1 , wherein the electrochemical signal of the electrochemically active marker is used to predict a response of an organism to a therapeutic or toxic agent.
46 .- 84 . (canceled)
85 . The method as claimed in claim 1 , in which two or more oligonucleotide probes are used, each probe being labeled with a different electrochemically active marker.
86 . The A method as claimed in claim 85 , in which the two or more electrochemically active markers have peaks in their voltammogram traces that are resolvable from each other.
87 . The method as claimed in claim 1 , wherein the electrochemically active marker is covalently linked to the oligonucleotide via a spacer comprising an aliphatic chain having 4 to 20 carbon atoms.
88 . The method as claimed in claim 87 , wherein the spacer is an aliphatic chain having 6 carbon atoms.
89 . The method as claimed in claim 1 , wherein the electrode is covered with a membrane.
90 . The method as claimed in claim 89 , wherein the membrane is able to selectively exclude molecules based on charge and size.
91 . The method as claimed in claim 1 , wherein the electrochemically active marker is at 2′, 3′, or 5′ position of a nucleotide within the oligonucleotide.