Methods and apparatus for detecting nucleic acid polymorphisms

• US 20010046673A1
• Filed: 01/25/2001
• Published: 11/29/2001
• Est. Priority Date: 03/16/1999
• Status: Abandoned Application

• Current Assignee: LJL Biosystems, Inc. (Danaher Corporation)
• Original Assignee: LJL Biosystems, Inc. (Danaher Corporation)
• Inventors: Richey, James S., Panfili, Peter R., Razvi, Enal S., Owicki, John C., Modlin, Douglas N., French, Todd E., Leytes, Lev J., Zhang-Klompus, Yan
• Application Number:
  US09/770,720
• Publication Number:
  US 20010046673A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

Methods and apparatus for detecting polynucleotide hybridization in luminescence-based assays. The methods may include (1) allele-specific hybridization, using luminescence detection, (2) allele-specific oligonucleotide ligation, using dye-labeled oligonucleotide ligation with luminescence resonance energy transfer (FRET) detection, and (3) allele-specific nucleotide incorporation, using primer extension with luminescence polarization (FP) detection. More specifically, the methods may include (1) locating a sample containing a nucleic acid material at an examination site, (2) illuminating the sample, (3) detecting light transmitted from the sample, and (4) determining the presence, absence, and/or identity of a nucleic acid target in the sample using the light transmitted from the sample.

Claims

1. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:

locating a sample containing a nucleic acid molecule at an examination site;

contacting the sample with a primer and at least one photoluminescent dideoxy terminator, where the primer binds specifically to a nucleic acid sequence adjacent to a polymorphic site in the nucleic acid molecule;

modifying the primer so that the polarization of light emitted by the terminator in response to illumination with polarized light is larger upon incorporation of the terminator into the modified primer than upon incorporation of the terminator into an unmodified primer;

illuminating the sample with polarized light capable of stimulating photoluminescence emission from the terminator;

detecting polarized light emitted from the sample sequence; and

determining the identity of an allele at the polymorphic site based on the extent of polarization of the light transmitted from the sample.

2. The method of

claim 1

, where the polymorphic site corresponds to a single nucleotide polymorphism.

3. The method of

claim 1

further comprising the step of amplifying the nucleic acid molecule prior to the step of locating the sample containing the nucleic acid at the examination site.

4. The method of

claim 3

, the primer being an extension primer, where the step of amplifying the nucleic acid comprises:

combining the nucleic acid with an amplification primer, a deoxynucleotide, and a polymerase;

performing one or more cycles of polymerase chain reaction to amplify the nucleic acid; and

removing any unused amplification primer and deoxynucleotide.

5. The method of

claim 1

, where the step of contacting the sample with a primer and a terminator comprises:

forming a complex between the primer and the nucleic acid molecule , where the primer is specifically bound to the nucleic acid molecule immediately upstream from the polymorphic site; and

incorporating the terminator onto the end of the primer using a polymerase, where the terminator is complementary to the nucleotide at the polymorphic site.

6. The method of

claim 1

, where the step of modifying the primer includes the step of positioning an energy transfer acceptor on the primer so that energy transfer will occur from the terminator to the acceptor upon incorporation of the terminator into the modified primer.

7. The method of

claim 1

, where the step of modifying the primer includes the step of positioning a quencher on the primer so that the quencher will reduce photoluminescence emission from the terminator upon incorporation of the terminator into the modified primer.

8. The method of

claim 1

, where the step of modifying the primer decreases the photoluminescence lifetime of the terminator upon incorporation of the terminator into the modified primer.

9. The method of

claim 1

, where the step of modifying the primer includes the step of binding a mass label to the primer to increase the effective mass of the primer.

10. The method of

claim 9

, where the step of binding the mass label to the primer includes the step of covalently attaching the mass label to the primer.

11. The method of

claim 9

, where the step of binding the mass label to the primer includes the step of forming a noncovalent complex between the mass label and the primer.

12. The method of

claim 9

, where the sample is positioned in a sample holder, and where the mass label is a surface of the sample holder.

13. The method of

claim 9

, where the mass label is a bead.

14. The method of

claim 1

, where the step of modifying the primer increases the rotational correlation time of the primer.

15. The method of

claim 1

, where the step of modifying the primer is performed before the step of contacting the sample with the primer and the terminator.

16. The method of

claim 1

, where the step of determining the identity of the allele includes the step of identifying the nucleotide present at the site of the polymorphism.

17. The method of

claim 1

, where the extent of polarization is assessed by computing at least one of a polarization and an anisotropy.

18. The method of

claim 1

, where the photoluminescence lifetime of the unincorporated terminator is greater than the rotational correlation time of the unincorporated terminator, and where the photoluminescence lifetime of the incorporated terminator is less than the rotational correlation time of the incorporated terminator.

19. The method of

claim 1

, the sample being a first sample, further comprising:

repeating with a second sample the steps of locating, contacting, modifying, illuminating, detecting, and determining; and

comparing the identities of the alleles in the first and second samples.

20. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:

locating a sample containing a nucleic acid molecule at an examination site;

contacting the sample with a primer and first and second photoluminescent dideoxy terminators, where the primer binds specifically to a nucleic acid sequence adjacent the polymorphic site, and where the first and second terminators have different photoluminescence lifetimes;

illuminating the sample with light capable of simultaneously stimulating photoluminescence emission from the first and second terminators;

detecting light emitted from the sample;

converting the detected light to a signal;

processing the signal based on the difference in lifetimes to discriminate between a first portion of the signal attributable to light emitted by the first terminator and a second portion of the signal attributable to light emitted by the second terminator; and

determining the identity of the allele at the polymorphic site based on a difference in the first and second portions.

21. The method of

claim 20

, where the polymorphic site corresponds to a single nucleotide polymorphism.

22. The method of

claim 20

, where the step of illuminating includes illuminating the sample with polarized light, and where the step of detecting includes detecting polarized light emitted from the sample.

23. The method of

claim 22

, where the difference in the first and second portions includes a difference in the extent of polarization of the light represented by the first and second portions.

24. The method of

claim 23

, where the extent of polarization is assessed by computing at least one of a polarization and an anisotropy.

25. The method of

claim 20

, where the step of processing includes processing the signal to further discriminate between a third portion of the signal attributable to background.

26. The method of

claim 20

, where the step of contacting includes contacting the sample with a third photoluminescent dideoxy terminator, and where the step of processing includes processing the signal to discriminate between a third portion of the signal attributable to light emitted by the third terminator.

27. The method of

claim 26

, where the first and second portions of the signal are discriminated from the third portion of the signal based on a difference in photoluminescence lifetime between the first, second, and third terminators.

28. The method of

claim 26

, where the first and second portions of the signal are discriminated from the third portion of the signal based on a difference in emission spectrum between the first and second terminators and the third terminator.

29. The method of

claim 20

, where the step of determining the identity of the allele includes the step of identifying the nucleotide present at the polymorphic site.

30. A method of detecting an allele at a polymorphic site in a nucleic acid sample comprising

packaging in stabilized form in a microplate well at least one reagent for performing a single base extension assay, where the reagent is selected from the group consisting of DNA polymerase, at least one ddNTP, and an extension primer capable of hybridizing to a known sequence in the nucleic acid sample adjacent the polymorphic site,

hydrating the reagent in the microplate well,

adding to the microplate well any additional reagents that are required to perform a single base extension assay,

contacting the hydrated reagent with a nucleic acid sample,

extending a primer that is hybridized to the nucleic acid sample by one terminating base corresponding to the polymorphic site, and

determining the allele at the polymorphic site and the nucleic acid sample based on the identity of the terminating base.

31. The method of

claim 30

, wherein the packaging step includes the step of substantially dehydrating the reagent in the microplate well.

32. The method of

claim 30

, wherein the packaging step includes the step of providing the reagent in a buffered concentrate.

33. The method of

claim 30

, wherein the packaging step includes the step of immobilizing the reagent on a solid support.

34. The method of

claim 30

, wherein the packaging step includes the step of immobilizing the reagent on a wall of the microplate well.

35. The method of

claim 30

, wherein the packaging step includes the step of providing at least two different ddNTPs, each ddNTP being labeled with a different luminophore.

36. The method of

claim 35

, wherein the ddNTPs are labeled with luminophores that emit at different wavelengths.

37. The method of

claim 35

, wherein the ddNTPs are labeled with luminophores that have different lifetimes.

38. The method of

claim 30

, wherein the packaging step includes the step of providing at least two different ddNTPs, DNA polymerase, and an extension primer capable of hybridizing to a known sequence in the nucleic acid sample adjacent the polymorphic site.

39. The method of

claim 30

, wherein the determining step includes the step of detecting polarization of a luminophore bound to a primer.

40. The method of

claim 30

, wherein the hydrating step is performed by the adding step.

41. The method of

claim 30

further comprising the step of amplifying the nucleic acid sample prior to the contacting step.

42. The method of

claim 30

, wherein the packaging step includes the step of providing different ddNTPs in different wells in a single microplate.

43. The method of

claim 30

, wherein the microplate well is one of a plurality of wells arranged in a density of at least about 4 wells per 81 mm².

44. The method of

claim 30

, wherein the microplate well has a maximum volume capacity of less than about 50 microliters.

45. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:

locating a sample containing a nucleic acid molecule at an examination site;

contacting the sample with a primer and a photoluminescent dideoxy terminator, where the primer binds specifically to a nucleic acid sequence adjacent a polymorphic site in the nucleic acid molecule;

directing excitation light from a surface toward the sample, where the excitation light is capable of stimulating photoluminescence emission from the terminator, and where the surface conveys substantially more than half of the incident excitation light;

directing emission light from the surface toward a detector, where the surface conveys substantially more than half of the incident emission light; and

determining the identity of the allele at the polymorphic site based on the emission light conveyed to the detector.

46. The method of

claim 45

, wherein the step of directing excitation light includes the step of reflecting excitation light off a surface toward the sample, and

wherein the step of directing emission light includes the step of transmitting emission light emitted from the sample through the surface toward the detector.

47. The method of

claim 46

, wherein the step of directing excitation light includes the step of transmitting excitation light through the surface toward the sample, and

wherein the step of directing emission light includes the step of reflecting emission light off the surface toward the sample.

48. The method of

claim 45

further comprising the step of positioning a dichroic mirror in the path of the excitation light and the emission light.

49. The method of

claim 48

, wherein the dichroic mirror is a multi-chroic mirror.

50. The method of

claim 45

, wherein the contacting step includes the step of contacting the sample with a plurality of dideoxy terminators, each dideoxy terminator being labeled with a different luminophore.

51. The method of

claim 50

, wherein the surface is provided on a multi-chroic mirror characterized by a separate transmission wavelength range and reflection wavelength range matched to each luminophore.

52. The method of

claim 51

, wherein the contacting step includes the step of providing four different photoluminescent dideoxy terminators, and providing a quadruple-chroic mirror in the path of the excitation light and the emission light.