Detection of polynucleotide hybridization

• US 20040241699A1
• Filed: 04/01/2004
• Published: 12/02/2004
• Est. Priority Date: 10/04/2001
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Zocchi, Mukta Singh, Zocchi, Giovanni
• Application Number:
  US10/491,353
• Publication Number:
  US 20040241699A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

The invention disclosed herein provides a new detection scheme to monitor hybridization between complimentary polynucleotides such as DNA and/or RNA. Embodiments of the invention disclosed herein localized electromagnetic radiation to provide an optimized analysis of polynucleotide hybridization in contexts such as the polynucleotide microarrays typically used on gene chips.

Claims

1. A method of detecting hybridization between a polynucleotide probe and a target polynucleotide having a nucleic acid sequence that is complementary to a nucleic acid sequence in the polynucleotide probe, wherein a first end of the polynucleotide probe is coupled to a matrix and a second end of the polynucleotide probe is coupled to a detectable marker, the method comprising observing a change in the conformation of the polynucleotide probe that is the result of hybridization between the polynucleotide probe and the target polynucleotide.

2. The method of claim 1, wherein the change in the conformation of the polynucleotide probe is observed by observing a change in the height of the detectable marker above the surface of the matrix that results from the hybridization between the polynucleotide probe and the target polynucleotide.

3. The method of claim 1, wherein the change in the conformation of the polynucleotide probe is observed by observing a stiffening of the probe that is the result of hybridization between the polynucleotide probe and the target polynucleotide.

4. The method of claim 2, wherein the change in the height of the detectable marker above the surface of the matrix is observed by evanescent wave scattering.

5. The method of claim 1, wherein the change in the conformation is correlated to the degree of complementarity between the polynucleotide probe and the target polynucleotide.

6. The method of claim 1, wherein the change in the conformation is correlated to the relative amounts of the polynucleotide probe and the target polynucleotide.

7. The method of claim 1, further comprising labelling the target polynucleotide with a detectable marker.

8. The method of claim 1, wherein the polynucleotide probe is about 30 to about 300 nucleotide residues in length.

9. The method of claim 1, wherein the matrix is a gene chip comprising a plurality of polynucleotide probes.

10. The method of claim 1, wherein the detectable marker is a fluorescent compound, a polymer bead or a light scattering particle.

11. The method of claim 1, further comprising creating a negative charge on the surface of the matrix by immobilizing negatively charged molecules on the surface of the matrix.

12. A method of detecting hybridization between a polynucleotide probe and a target polynucleotide having a nucleic acid sequence that is complementary to a nucleic acid sequence in the polynucleotide probe, wherein the polynucleotide probe has a first end labeled with a detectable marker and a second end attached to a matrix having a negative charge, the method comprising using evanescent wave illumination to observe a reduction in the height of a detectable marker coupled to the polynucleotide probe'"'"'s free end above the surface of the matrix to which the polynucleotide probe is attached.

13. The method of claim 12, wherein the detectable marker is a fluorescent compound or a light scattering particle.

14. The method of claim 12, wherein the target polynucleotide is not labelled with a detectable marker.

15. The method of claim 12, wherein the matrix is a gene chip comprising a plurality of polynucleotide probes.

16. A method of detecting hybridization between a polynucleotide probe and a target polynucleotide having a nucleic acid sequence that is complementary to a nucleic acid sequence in the polynucleotide probe, wherein the polynucleotide probe has a bound end coupled to a matrix and a free end coupled to a detectable marker, the method comprising:

(a) determining an height of the detectable marker coupled to the polynucleotide probe'"'"'s free end above the surface of the matrix to which the probe is attached in the absence of a complementary polynucleotide sequence;

(b) allowing the polynucleotide probe and the target polynucleotide sequence to come into contact with one another under conditions favorable to hybridization;

(c) using evanescent wave illumination to measure the height of the detectable marker coupled to the polynucleotide probe'"'"'s free end above the surface of the matrix to which the probe is attached in the presence of the target polynucleotide sequence;

(d) comparing the height of the detectable marker in step (a) with the height of the detectable marker in step (c);

wherein a reduction the height of the detectable marker in step (a) as compared to step (d) is indicative of hybridization between a polynucleotide probe and a target polynucleotide having a nucleic acid sequence that is complementary to a nucleic acid sequence in the polynucleotide probe.

17. An apparatus for detecting hybridization between a polynucleotide probe and a target polynucleotide having a nucleic acid sequence that is complementary to a nucleic acid sequence in the polynucleotide probe, wherein the hybridization is detected using evanescent wave illumination, the apparatus comprising:

(a) a matrix on which a first end of a polynucleotide probe attached, wherein the second end of the polynucleotide probe is coupled to a detectable marker consisting of a fluorophore or a light scattering marker;

(b) a coupling mechanism which optically couples the probe to an optical guide to obtain an evanescent wave on the surface of the matrix;

(c) an optical arrangement which measures the fluorescent or scattered intensity both before and after depositing a solution containing a target polynucleotide sequences on the probe under conditions which favor hybridization of the probe and a target polynucleotide sequences that are complementary to a nucleic acid sequence in the polynucleotide probe; and

(d) a detector which records the difference of fluorescent intensity or scattering before and after subjecting the probe DNA to the target polynucleotide sequences.

18. A kit comprising a container, a label on said container, and a polynucleotide probe composition contained within said container;

wherein a first end of the polynucleotide probe is coupled to a matrix and a second end of the polynucleotide probe is coupled to a detectable marker; and

instructions for using the polynucleotide probe composition in methods of detecting hybridization between a polynucleotide probe and a target polynucleotide having a nucleic acid sequence that is complementary to a nucleic acid sequence in the polynucleotide probe by observing a change in the conformation of the polynucleotide probe that is the result of hybridization between the polynucleotide probe and the target polynucleotide.

19. The kit of claim 18, wherein the detectable marker is selected to be compatible for use with evanescent wave illumination.

20. The kit of claim 19, wherein the matrix is a gene chip and further wherein the surface of the gene chip is negatively charged.