Nucleic acid reactions using labels with different redox potentials

• US 20030143556A1
• Filed: 04/30/2002
• Published: 07/31/2003
• Est. Priority Date: 04/03/2001
• Status: Abandoned Application

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Kayyem, Jon Faiz, Tao, Chunlin, Blackburn, Gary, Yu, Changjun
• Application Number:
  US10/137,710
• Publication Number:
  US 20030143556A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention is directed to methods and compositions for the use of electron transfer moieties with different redox potentials to electronically detect nucleic acids, particularly for the electrochemical sequencing of DNA.

Claims

1. A composition comprising:

a) a first nucleic acid comprising a first ETM with a first redox potential; and

b) a second nucleic acid comprising a second ETM with a second redox potential;

c) a third nucleic acid comprising a third ETM with a third redox potential; and

d) a fourth nucleic acid comprising a fourth ETM with a fourth redox potential, wherein said first, second, third and fourth redox potentials are different.

2. A composition according to claim 1 wherein the sequences of said first, second, third and fourth nucleic acids are different.

3. A composition according to claim 2 wherein the sequence of first, second, third and fourth nucleic acids differ by only one base.

4. A composition according to claim 3 wherein the nucleoside comprising the different base comprises said ETM.

5. A composition according to claim 1 wherein said nucleic acids are single stranded.

6. A composition according to claim 1 wherein at least one of said ETMs is a transition metal complex.

7. A composition according to claim 6 wherein said transition metal complex is ferrocene.

8. A composition according to claim 1 wherein all of said ETMs are ferrocene derivatives. S

9. A composition according to claim 6 wherein at least one of said transition metal complexes is a ruthenium complex.

10. A composition according to claim 6 wherein all of said ETMs are ruthenium derivatives.

11. A method of determining the identification of a nucleotide at a detection position in a target sequence, wherein said target sequence comprises a first target domain directly 5′

adjacent to said detection position, said method comprising;

a) providing a first hybridization complex comprising said target sequence and an extension primer hybridized to said first target domain of said target sequence;

b) contacting said hybridization complex with;

i) a polymerase enzyme; and

ii) a composition comprising a plurality of chain terminating NTPs each comprising a covalently attached ETM, each NTP comprising an ETM with a different redox potential;

under conditions whereby if one of said NTPs basepairs with the base at said detection position, said extension primer is extended by said enzyme to incorporate said ETM and form an extended primer; and

c) identifying the base at said detection position.

12. A method according to claim 11 wherein said identification step comprises:

a) contacting said extended primer with a solid support comprising an array of electrodes comprising capture probes to form second hybridization complexes;

b) applying an input signal to said electrodes; and

c) detecting an output signal characteristic of said ETM.

13. A method according to claim 11 wherein said extension primer is attached to an electrode on a solid support.

14. A method according to claim 13 wherein said identification step comprises:

a) applying an input signal to said electrodes; and

b) detecting an output signal characteristic of said ETM

15. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:

a) providing a solid support comprising an array of electrodes each comprising a capture probe;

b) contacting said array with a plurality of detection probes each comprising;

i) a unique nucleotide at the interrogation position; and

ii) an ETM with a unique redox potential; and

c) detecting a signal from at least one of said ETMs to identify the nucleotide at the detection position.

16. A method of sequencing a target nucleic acid comprising:

a) providing a plurality of sequencing probes complementary to said target sequence, each of a different length, each comprising a different chain terminating NTP comprising an ETM comprising a different redox potential;

b) separating said nucleic acids on the basis of size; and

c) detecting each of said ETMs to identify the sequence of at least a portion of said target nucleic acid.

17. A method of making a plurality of sequencing probes, each with a covalently attached ETM with a different redox potential, said method comprising:

a) providing a first oligonucleotide substituted with a first 5′

protected deoxynucleotide;

b) providing a first ETM derivative with a first redox potential;

c) mixing said first oligonucleotide with said first ETM derivative to form a first sequencing probe with a first deoxynucleotide triphosphate comprising a first ETM with a first redox potential;

d) providing a second oligonucleotide substituted with a second 5′

protected deoxynucleotide;

e) providing a second ETM derivative with a second redox potential;

f) mixing said second oligonucleotide with said second ETM derivative to form a second sequencing probe with a second deoxynucleotide triphosphate comprising a second ETM with a second redox potential

18. A method according to claim 17 further comprising:

a) providing a third oligonucleotide substituted with a third 5′

protected deoxynucleotide;

b) providing a third ETM derivative with a third redox potential; and

c) mixing said third oligonucleotide with said third ETM derivative to form a third sequencing probe with a third deoxynucleotide triphosphate comprising a third ETM with a third redox potential.

19. A method according to claim 18 further comprising:

a) providing a fourth oligonucleotide substituted with a fourth 5′

protected deoxynucleotide;

b) providing a fourth ETM derivative with a fourth redox potential; and

c) mixing said fourth oligonucleotide with said fourth ETM derivative to form a fourth sequencing probe with a fourth deoxynucleotide triphosphate comprising a fourth ETM with a fourth redox potential.

20. A method according to claims 17, 18, and 19 wherein said first, second, third and fourth deoxynucleotide triphosphates are different.

21. A composition according to claims 17, 18, and 19 wherein at least one of said ETMs is a transition metal complex.

22. A composition according to claim 21 wherein said transition metal complex is ferrocene.

23. A method according to claim 21 wherein said detecting comprises passing said sequencing probes over four sequential electrodes comprising different potentials.

24. A method according to claim 21 wherein said detecting comprises passing said sequencing probes over a single electrode.

25. A method of making a plurality of nucleic acids, each with a covalently attached ETM with a different redox potential, said method comprising:

a) providing a first transitional metal complex with a first redox potential and a first functional group;

b) providing a first oligonucleotide substituted with a second functional group; and

c) mixing said first transition metal complex with said first oligonucleotide to form a first transition metal complex-oligonucleotide conjugate with a first redox potential;

d) providing a second transitional metal complex with a second redox potential and a first functional group;

b) providing a second oligonucleotide substituted with a second functional group; and

c) mixing said second transition metal complex with said second oligonucleotide to form a second transition metal complex-oligonucleotide conjugate with a second redox potential.