Assays and apparatus for detecting electrochemical active markers in an electric field
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.
1. A method of detecting a target nucleic acid sequence in a nucleic acid solution, comprising:
contacting the nucleic acid solution with one or more oligonucleotide probes each labeled with a metallocene moiety, wherein each oligonucleotide probe is covalently linked to a metallocene moiety;
providing conditions at which one or more of the oligonucleotide probes hybridize with a complementary target sequence present in the nucleic acid solution and form a double stranded hybridized nucleic acid, wherein each of the one or more oligonucleotide probes is complementary along its length to the target sequence;
degrading the double stranded hybridized nucleic acid by digestion with a duplex specific nuclease to form at least one of a mononucleotide degraded probe labeled with the metallocene moiety and a dinucleotide degraded probe labeled with the metallocene moiety, wherein the duplex specific nuclease is capable of degrading the oligonucleotide probe only when the oligonucleotide probe is present in the double stranded hybridized nucleic acid and, in the presence of the double stranded hybridized nucleic acid, the duplex specific nuclease degrades the oligonucleotide probe to form at least one of a mononucleotide degraded probe labeled with the metallocene moiety and a dinucleotide degraded probe labeled with the metallocene moiety; and
electrochemically detecting a signal based on redox characteristics of the mononucleotide probe labeled with the metallocene moiety, the dinucleotide degraded probe labeled with the metallocene moiety, and any unhybridized oligonucleotide probe in the solution, wherein the signal is influenced by at least the size of the probe to which the metallocene moiety is attached; and wherein the signal of the mononucleotide probe or the dinucleotide probe detects the target nucleic acid sequence in the nucleic acid solution.
2. The method of claim 1 , wherein each of the one or more oligonucleotide probes is covalently linked to a metallocene moiety via a spacer comprising an aliphatic chain having 4 to 20 carbon atoms.
3. The method according to claim 1 , wherein detecting the target sequence is used for the detection of nucleic acid polymorphisms.
4. The method according to claim 1 , wherein the method detects an amplified target nucleic acid in the solution.
5. The method according to claim 1 , wherein a single metallocene moiety is attached to the 5′-terminal nucleotide of each of the one or more oligonucleotide probes.
6. The method according to claim 1 , wherein a single metallocene moiety is attached to the 3′-terminal nucleotide of each of the one or more oligonucleotide probes.
7. The method according to claim 1 , wherein multiple metallocene moieties are attached along the length of each of the one or more oligonucleotide probes.
8. The method of claim 1 , wherein the duplex specific nuclease is T7 exonuclease.
9. The method of claim 1 , wherein the metallocene moiety is a ferrocene moiety.
10. The method according to claim 2 , wherein the spacer is an aliphatic chain having 6 carbon atoms.
11. The method of claim 1 , wherein detecting the target sequence is used for detection of allelic polymorphisms.
12. The method of claim 1 , wherein detecting the target sequence is used for the detection of single nucleotide polymorphisms.
13. The method of claim 1 , wherein detecting the target sequence is used for the quantification of nucleic acid species.
14. The method of claim 1 , wherein detecting the target sequence is used for the quantification of gene expression.
15. The method of claim 1 , wherein electrochemically detecting the signal of the mononucleotide probe, the dinucleotide probe, and any unhybridized oligonucleotide probe is by voltammetry.
16. The method of claim 1 , wherein electrochemically detecting the signal of the mononucleotide probe, the dinucleotide probe, and any unhybridized oligonucleotide probe is by an amperometric technique.
17. The method of claim 1 , wherein electrochemically detecting the signal of the mononucleotide probe, the dinucleotide probe, and any unhybridized oligonucleotide probe is by differential pulse voltammetry.
18. The method of claim 9 , wherein the ferrocene moiety is a di-ferrocene moiety.
19. The method according to claim 9 , wherein multiple oligonucleotide probes are each labeled with different derivatives of ferrocene.
20. The method according to claim 19 , wherein the different derivatives of ferrocene have peaks at distinct voltages.