Methods and apparatus for detecting polynucleotide hybridization

• US 6,503,719 B2
• Filed: 01/22/2001
• Issued: 01/07/2003
• Est. Priority Date: 01/15/1999
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: LJL Biosystems, Inc. (Danaher Corporation)
• Inventors: Owicki, John C., Modlin, Douglas N., French, Todd E., Petersen, Jon F.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/767,316
• Publication Number:
  US 20010034025A1
• Time in Patent Office: 715 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/0405 manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
• CPC Class Codes: G01N 2035/0425 Stacks, magazines or elevators for plates
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1011 Control of the position or alignment of the transfer device
• CPC Class Codes: G01N 35/1074 arranged in a two-dimensional array
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods and apparatus for detecting polynucleotide hybridization in luminescence-based assays. The methods may include (1) contacting a sample polynucleotide with a reference polynucleotide at an assay site, where at least one of the polynucleotides is capable of emitting luminescence, (2) illuminating the assay site with light capable of stimulating such luminescence, (3) detecting light transmitted from the assay site, and (4) deriving information relating to the extent of hybridization between the sample and reference polynucleotides based on the detected light. The methods may further include (1) illuminating with and/or detecting polarized light, (2) deriving information relating to the sequence of the sample polynucleotide from the extent of hybridization, and (3) converting the light to a signal and distinguishing between a portion of the signal attributable to luminescence and a portion attributable to background.

Claims

1. A method of detecting a polynucleotide, the method comprising:

providing a substrate having at least two assay sites;

locating a different reference polynucleotide at each assay site;

depositing substantially equal amounts of a sample polynucleotide at each assay site under conditions conducive to hybridization, wherein at least one of the reference and sample polynucleotides at each assay site is labeled with a luminophore;

illuminating each assay site with polarized light;

detecting polarized light transmitted from each assay site; and

deriving information relating to the sequence of the sample polynucleotide by comparing the extent of polarization of the light emitted from each assay site.

2. The method of claim 1, wherein the substrate is a microplate having a plurality of wells, and wherein each assay site is located in a different one of the wells.

3. The method of claim 2, wherein each well has a frusto-conical shape.

4. The method of claim 2, wherein the wells are organized in a density of at least about 4 wells per 81 mm².

5. The method of claim 2, wherein each well has a maximum volume capacity of less than about 55 microliters.

6. The method of claim 1, wherein the substrate is a chip.

7. The method of claim 1, wherein the reference polynucleotides are bound to the substrate at each assay site.

8. The method of claim 1, wherein the reference polynucleotides are in solution at each assay site.

9. The method of claim 1, wherein the reference polynucleotides are selected from the group consisting of DNA, RNA, and PNA.

10. The method of claim 1, wherein the reference polynucleotides are oligonucleotides.

11. The method of claim 1 further comprising:

amplifying the sample polynucleotide using the polymerase chain reaction prior to the step of depositing the sample polynucleotide at each assay site.

12. The method of claim 1, wherein the sample polynucleotides are selected from the group consisting of DNA, RNA, and PNA.

13. The method of claim 1, wherein the sample polynucleotides are labeled with the luminophore.

14. The method of claim 1, wherein the reference polynucleotides are labeled with the luminophore.

15. The method of claim 1 further comprising:

permitting the sample and reference polynucleotides to hybridize prior to the step of illuminating each assay site.

16. The method of claim 1 further comprising:

forming a complex between the sample polynucleotide, the reference polynucleotide, and a mass label prior to the step of illuminating each assay site.

17. The method of claim 1, wherein the concentration of reference polynucleotide is sufficient relative to the concentration of sample polynucleotide to produce detectable hybridization between the reference and sample polynucleotides.

18. The method of claim 1, the reference polynucleotides being double-stranded, further comprising the step of treating the reference polynucleotides to increase binding by the sample polynucleotides.

19. The method of claim 1, wherein the step of deriving information relating to the sequence includes the step of assaying for single nucleotide polymorphisms.

20. The method of claim 1, wherein the steps of illuminating and detecting are performed on a site-by-site basis.

21. The method of claim 1, wherein the steps of illuminating and detecting are performed on at least two sites simultaneously.

22. The method of claim 1, wherein the extent of polarization is assessed by determining a polarization or an anisotropy.

23. The method of claim 1 further comprising:

converting the light detected from each assay site to a signal; and

discriminating between a first portion of the signal that is attributable to light emitted by the luminophore and a second portion of the signal that is attributable to a background.

24. The method of claim 23, wherein the step of discriminating is performed without requiring a determination of the lifetime or intensity of the background.

25. The method of claim 23, wherein the step of discriminating is performed without requiring use of information obtained from a blank, irrespective of whether a significant amount of the background is being detected by the detector at the same time that light emitted by the analyte is being detected.

26. The method of claim 23, wherein the step of discriminating is performed in the frequency-domain without requiring a determination of the intensity of the background.

27. The method of claim 23, wherein the step of discriminating is performed in the frequency-domain without requiring use of information obtained from a blank.

28. The method of claim 23, wherein the background is attributable to the substrate.

29. A method of detecting a polynucleotide, the method comprising:

contacting a sample polynucleotide with a reference polynucleotide at an assay site, wherein at least one of the reference and sample polynucleotides is labeled with a luminophore;

illuminating the assay site with light capable of stimulating luminescence from the luminophore;

detecting light transmitted from the assay site;

converting the detected light to a signal;

discriminating between a first portion of the signal that is attributable to light emitted by the luminophore and a second portion of the signal that is attributable to a background; and

deriving information relating to the extent of hybridization between the sample polynucleotide and the reference polynucleotide based on the extent of polarization of the light emitted from the hybrid.

30. The method of claim 29, wherein the discriminating step uses lifetime-resolved methods.

31. The method of claim 30, wherein the discriminating step uses frequency-domain methods.

32. The method of claim 29 further comprising:

performing each of the steps at a second assay site, wherein the reference polynucleotide is different at the two sites and the sample polynucleotide is the same at the two sites.

33. The method of claim 32 further comprising:

obtaining information concerning the sequence of the sample by comparing the extent of hybridization between the sample polynucleotide and the reference polynucleotide at each site.

34. The method of claim 29, wherein the discriminating step is performed without using information obtained from a blank.

35. The method of claim 29 further comprising:

combining a luminescent reference compound with the sample polynucleotide and the reference polynucleotide; and

determining the intensity of light emitted from the luminophore as a function of the intensity of light emitted from the reference compound.

36. The method of claim 35, wherein the determining step uses lifetime-resolved methods.

37. The method of claim 35 further comprising:

calculating a ratio of the intensity of light emitted from the luminophore to the intensity of light emitted from the reference compound.

38. A method of detecting hybridization between first and second polynucleotides, the method comprising:

providing a substrate containing at least two assay sites, each assay site being located in a well having a shape;

contacting a first polynucleotide with a second polynucleotide at each assay site, wherein at least one of the two polynucleotides is bound to a luminophore;

directing a light beam to each assay site, wherein the light beam has a shape that substantially matches the shape of the well; and

determining the extent of hybridization between the first polynucleotide and the second polynucleotide based on detecting polarized light emitted from the luminophore at each assay site.