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 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 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.