Sequence determination of nucleic acids using electronic detection
The present invention is directed to methods and compositions for the use of self-assembled monolayers to electronically detect nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms (SNPs).
1. A method of determining the identification of nucleotide(s) at a first detection position in a first domain of a target sequence, said target sequence comprising said first domain and a second domain, said method comprising:
a. providing an electrode with a covalently attached capture probe, wherein said capture probe has a sequence substantially complementary to said second domain of said target sequence;
b. contacting said electrode with:
(i) said target sequence;
(ii) a first label probe substantially complementary to said first domain, comprising a first nucleotide at an interrogation position and a first electron transfer moiety (ETM) with a first redox potential;
(iii) a second label probe substantially complementary to said first domain, comprising a second nucleotide at said interrogation position and a second ETM with a second redox potential;
under conditions wherein if said nucleotide at said interrogation position is perfectly complementary to said detection position, hybridization of said probe(s) occurs; and
c. detecting the presence of said first and/or second ETM to determine the nucleotide(s) at said detection position.
2. The method of claim 1 wherein said method further comprises contacting said electrode with a third label probe substantially complementary to said first domain, comprising a third nucleotide at said interrogation position and a third ETM with a third redox potential.
3. The method of claim 2 wherein said method further comprises contacting said electrode with a fourth label probe substantially complementary to said first domain, comprising a fourth nucleotide at said interrogation position and a fourth ETM with a fourth redox potential.
4. The method of claim 1 wherein said electrode comprises an array of capture probes, each substantially complementary to a second domain of a different target sequence.
5. The method of claim 1 wherein said first label probe contains a plurality of first ETMs.
6. The method of claim 1 wherein said second label probe contains a plurality of second ETMs.
7. The method of claim 1 wherein said electron transfer moieties comprise a transition metal complex.
8. The method of claim 7 wherein said transition metal complex comprises a metallocene.
9. The method of claim 8 wherein said metallocene is a ferrocene.
10. The method of claim 8 wherein said metallocene is a ferrocene derivative.