Single base extension

• US 20050153320A1
• Filed: 11/08/2004
• Published: 07/14/2005
• Est. Priority Date: 11/06/2003
• Status: Abandoned Application

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Tolley, Samuel, Herron, James N.
• Application Number:
  US10/984,629
• Publication Number:
  US 20050153320A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Genetic polymorphisms can change protein structure and function, altering dispositions to diseases and conditions. A single nucleotide polymorphism is the smallest genetic mutation and the most difficult to detect. However, single nucleotide polymorphisms also make up 90% of known genetic mutations, thus identifying such polymorphisms is essential. Single base extension uses the affinity of one base for its complementary base to detect polymorphisms, including single nucleotide polymorphisms. Planar waveguides are used as the platform for single base extension enabling rapid, real time detection of genetic polymorphisms. Detection limits in the picomolar range can be obtained. Signals from the non-matched DNA bases are in the range of the blank signal. Detection times of 5 minutes are reported.

Claims

1. A method of detecting a genetic polymorphism, the method comprising:

attaching a capture molecule to a solid support in at least one solution channel, wherein the at least one solution channel is in a flowcell and the solution channel is in close proximity with a waveguide;

injecting a sample, a polymerase, and a fluorescently labeled nucleotide into the at least one solution channel, under conditions wherein a single stranded analyte molecule present in the sample is capable of hybridizing to the capture molecule;

hybridizing the single stranded analyte molecule to the capture molecule;

extending the hybridized capture molecule by covalently adding a fluorescently labeled nucleotide to the hybridized capture molecule; and

detecting the presence or absence of the covalently added fluorescently labeled nucleotide.

2. The method according to claim 1, wherein the attaching a capture molecule to a solid support in at least one solution channel comprises attaching a plurality of different capture molecules patterned thereon to simultaneously detect different possible polymorphisms.

3. The method according to claim 1, wherein injecting the sample, the polymerase, and the nucleotide into the at least one solution channel comprises injecting at least two different fluorescently labeled nucleotides.

4. The method according to claim 3, wherein injecting the sample into the at least one solution channel comprises injecting the sample into at least two solution channels.

5. The method according to claim 1, wherein injecting the sample, the polymerase, and the nucleotide comprises injecting the polymerase and the sample prior to injecting the nucleotide.

6. The method according to claim 1, wherein the fluorescently labeled nucleotide comprises a Cy5-labeled dideoxynuclotide triphosphate.

7. The method according to claim 1, wherein the polymerase comprises thermostable DNA polymerase.

8. The method according to claim 7, wherein extending the hybridized capture molecule by covalently adding at least one fluorescently labeled nucleotide to the hybridized capture molecule is conducted at a temperature between about 40°

C. and a about 50°

C.

9. The method according to claim 8, wherein extending the hybridized capture molecule by covalently adding at least one fluorescently labeled nucleotide to the hybridized capture molecule is conducted at a pH between about pH 6 and about pH 8.5.

10. The method according to claim 1, wherein detecting the presence or absence of the covalently added fluorescently labeled nucleotide comprises detecting without washing the labeled nucleotide from the solution chamber.

11. The method according to claim 1, wherein extending the hybridized capture molecule by covalently adding at least one fluorescently labeled nucleotide to the hybridized capture molecule is conducted at a pH between about pH 6 and about pH 8.5.

12. The method according to claim 1, wherein detecting the presence or absence of the covalently added fluorescent nucleotide comprises using a CCD camera.

13. The method according to claim 12, wherein using the CCD camera comprises collecting data at 10-second intervals.

14. The method according to claim 13, wherein collecting data at 10-second intervals comprises collecting the data over a 5-minute period.

15. The method according to claim 14, wherein the flowcell comprises three solution channels, each in contact with approximately one-third of the waveguide.

16. The method according to claim 1, wherein injecting a sample, a polymerase, and a fluorescently labeled nucleotide into the at least one solution channel, under conditions wherein a single stranded analyte molecule present in the sample is capable of hybridizing to the capture molecule comprises injecting at least four labeled nucleotides, each nucleotide having a different fluorescent label, and utilizing multiplexing to simultaneously detect the addition of all four nucleotide possibilities in a single solution channel.

25. The assay of any of the preceding claims wherein the waveguide platform has different capture molecules patterned thereon so as to simultaneously detect different possible polymorphisms.

26. The assay of any of the preceding claims wherein the SBEX assay utilizes multiplexing to simultaneously detect all base possibilities in a single channel.

17. An assay system to detect a single nucleotide polymorphism, said assay system comprising:

a capture molecule attached to a solid support in contact with a solution channel, wherein the solution channel is in close proximity with a planar waveguide fluorescent biosensor;

a fluorescence biosensor capable of detecting a single base extension, wherein a single base extension comprises covalently attaching a fluorescently labeled nucleotide by the action of a polymerase to the capture molecule when a single stranded analyte molecule present in a sample is hybridized to the capture molecule.

18. The assay system of claim 17, comprising a wash-less assay system.

19. The assay system of claim 18, wherein the assay system utilizes multiplexing to simultaneously detect all base possibilities in a single solution channel.

20. An enzyme-catalyzed single base extension reaction used to detect single nucleotide polymorphisms with planar waveguide fluorescence biosensor technology.

21. A method of detecting genetic polymorphisms comprising using planar waveguides as the platform for single base extension.

22. An assay system to detect single nucleotide polymorphisms, said assay system comprising, in combination, a single base extension together with a planar waveguide fluorescent biosensor.

23. An enzyme-catalyzed single base extension reaction used to detect single nucleotide polymorphisms using planar waveguide fluorescence biosensor technology.

24. A wash-less assay comprising, in combination, a single base extension together with a planar waveguide.

27. An improved method of conducting a diagnosis using a waveguide, the improvement comprising:

conducting a fluorescent affinity type assay, single base extension (SBEX), on said waveguide.