Enzymatic ligation-based identification of transcript expression

• US 20030082584A1
• Filed: 06/28/2002
• Published: 05/01/2003
• Est. Priority Date: 06/29/2001
• Status: Abandoned Application

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Wang, Xun, Shi, Liang, Li, Bi-Yu
• Application Number:
  US10/187,039
• Publication Number:
  US 20030082584A1
• 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 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

Disclosed herein are novel methods for use in the analysis of nucleic acids. Uses disclosed include polynucleotide expression analysis and detection of single nucleotide polymorphisms. Also provided are diagnostic methods and kits for conducting the disclosed methods.

Claims

1. A method for determining polynucleotide expression comprising:

providing a population of polynucleotides, wherein said population comprises at least one target polynucleotide comprising a known nucleotide sequence;

providing for each target polynucleotide, a pair of specific sensor probes, each probe in said pair having a 3′

end and a 5′

end, a first probe of said pair having a 3′

portion that is complementary to said target polynucleotide and a 5′

portion comprising a common primer binding site, and a second probe of said pair having a 5′

portion complementary to said target polynucleotide and a 3′

portion comprising a common primer binding site, wherein said complementary portions on said sensor probes are immediately adjacent on said target polynucleotide;

combining said at least one polynucleotide target with its pair of specific sensor probes under stringent hybridization conditions;

allowing said sensor probes to hybridize to said at least one target polynucleotide;

ligating hybridized members of a pair of sensor probes to form a ligated sensor probe;

amplifying said ligated sensor probes to provide amplified ligated sensor probes wherein said amplified ligated sensor probes comprise a detectable label;

for each different pair of sensor probes providing at least one class of detector oligonucleotide, said detector oligonucleotide comprising a detectable label that is different for each class of detector oligonucleotide and capable of being differentiated from the label of said amplified ligated sensor probes, wherein said detector oligonucleotide is capable of hybridizing to a portion of said ligated sensor probes that is complementary to said target polynucleotide;

combining said labeled amplified ligated sensor probes with said detector oligonucleotides under stringent conditions and allowing said detector oligonucleotides to hybridize to said amplified ligated sensor probes;

determining the hybridization of said detector oligonucleotides to said amplified ligated sensor probes by detecting the presence of said detectable label of said detector oligonucleotide in association with said detectable label of said amplified ligated sensor probes; and

identifying said target polynucleotide by the identity of the detector oligonucleotide.

2. The method of claim 1, wherein said detector oliognucleotide further comprises a microsphere, said microsphere comprising said detectable label for said detector oligonucleotide.

3. The method of claim 2, wherein said microbead is a Luminex microsphere.

4. The method of claim 1, wherein said detectable labels are detected by a flow cytometer.

5. The method of claim 1, wherein said determination of said detectable labels is quantitative.

6. The method of claim 1, wherein said ligation is achieved using a T4 ligase.

7. The method of claim 1, wherein said at least one target polynucleotide is obtained from a plant or an animal.

8. The method of claim 1, wherein said at least one target polynucleotide comprises DNA

9. The method of claim 1, wherein said at least one target polynucleotide comprises RNA.

10. The method of claim 1, wherein said detector oligonucleotides hybridizes to a portion of said ligated sensor probes that comprises said 3′

end of said first sensor probe and said 5′

end of said second sensor probe.

11. The method of claim 1, wherein said amplification uses primers that bind to said common primer binding sites.

12. The method of claim 11, wherein said amplification is by the polymerase chain reaction.

13. The method of claim 1, wherein said population of polynucleotides comprises at least 20 different target polynucleotides.

14. The method of claim 1, wherein said population of polynucleotides comprises at least 50 different target polynucleotides.

15. The method of claim 1, wherein said population of polynucleotides comprises at least 100 different target polynucleotides.

16. The method of claim 1, wherein said detector oligonucleotides are from about 18 nucleotides long to about 30 nucletotides long.

17. The method of claim 1, wherein said portion of each sensor probe in a sensor probe pair that is complementary to the target polynucleotide is from about 15 nucleotides long to about 30 nucleotides long.

18. The method of claim 17, wherein said portion of each sensor probe in a sensor probe pair that is complementary to the target polynucleotide is about 20 nucleotides long.

19. The method of claim 1, wherein said common primer binding site is from about 12 nucleotides long to about 24 nucleotides long.

20. The method of claim 19, wherein said common primer site is about 18 nucleotides long.

21. The method of claim 1, wherein each of said sensor probes comprise a first portion complementary to said target polynucleotide and said detector oligonucleotide;

a second portion complementary to said target polynucleotide, but not complementary to said detector oligonucleotide; and

a third portion comprising a common primer binding site that is not complementary to either said target polynucleotide or said detector polynucleotide.

22. The method of claim 21, wherein when hybridized to said target polynucleotide, said first portions of each of the sensor probes in a sensor probe pair are adjacent to each other.

46. A method for determining the physiological or developmental state of a cell or tissue comprising,

obtaining a population of polynucleotides from a test cell or test tissue of an unknown physiological or developmental state, wherein said population comprises at least one target polynucleotide of interest;

determining expression of said at least one target polynucleotide of interest by the method of claim 1;

and

comparing expression of said at least one target polynucleotide from said test cell or tissue to expression of said at least one target polynucleotide obtained from a reference cell or tissue of a known physiological or developmental state also determined by the method of claim 1.

47. A method of diagnosing a disease, condition, disorder, or predisposition comprising:

obtaining a population of polynucletotides from a cell or tissue of a test subject, wherein said population comprises at least one target polynucleotide of interest;

determining expression of said at least one target polynucleotide of interest by the method of claim 1;

and

comparing expression of said at least one target polynucleotide of interest from said subject to the expression of said at least one target polynucleotide of interest obtained from a reference subject known to have said disease, condition, disorder, or predisposition also determined by the method of claim 1.

50. A kit, comprising at least one pair of sensor probes for at least one target polynucleotide, at least one detector oligonucleotide for each pair of sensor probes, and instructions for carrying out the method of claim 1.

51. The kit of claim 50, wherein said at least one detector oligonucleotide further comprises a labeled microbead or microsphere.

23. A method for detecting a single nucleotide polymorphism comprising:

providing a population of polynucleotides, wherein said population comprises at least one target polynucletotide containing a single nucleotide polymorphism;

providing for each target polynucleotide at least one pair of specific sensor probes, each probe in said pair having a 3′

end and a 5′

end, a first sensor probe of said pair having a 3′

portion that is complementary to said target polynucleotide and a 5′

portion comprising a common primer binding site, and a second sensor probe of each pair having a 5′

portion that is complementary to said target polynucleotide and a 3′

portion comprising a common primer binding site, wherein said complementary portions on said sensor probes in said pair are immediately adjacent on said target polynucleotide and either the 3′

end of said first probe or the 5′

end of said second probe is complementary to an allele of said single nucleotide polymorphism;

combining said at least one polynucleotide target with its at least one pair of specific sensor probes under stringent hybridization conditions;

allowing said sensor probes to hybridize to said at least one target polynucleotide;

ligating hybridized members of a pair of sensor probes to form a ligated sensor probe under conditions such that if the single nucleotide polymorphism allele present is not complementary to said sensor probes then ligation does not occur;

amplifying said ligated sensor probes to provide amplified ligated sensor probes wherein said amplified ligated sensor probes comprise a detectable label;

for each pair of different sensor probes providing at least one class of detector oligonucleotide, said oliognucleotides comprising a detectable label that is different for each class of detector oligonucleotide and capable of being differentiated from the label of said amplified ligated sensor probes, wherein said detector oligonucleotides are capable of hybridizing to a portion of said ligated sensor probe that is complementary to said target polynucleotide;

combining said amplified ligated sensor probes with said detector oligonucleotides under stringent conditions and allowing said detector oligonucleotides to hybridize to said amplified ligated sensor probes;

determining the hybridization of said detector oligonucleotides to said amplified ligated sensor probes by detecting the presence of said detectable label of said detector oligonucleotides in association with said detectable label of said amplified ligated sensor probes; and

determining the presence, absence or frequency of said allele of said single nucleotide polymorphism, said allele identified by the detector oligonucleotide label.

24. The method of claim 23 further comprising, providing for each target polynucleotide at least a first and a second pair of specific sensor probes wherein either the 3′

end of said first probe or the 5′

end of said second probe of said first pair of sensor probes is complementary to an allele of said single nucleotide polymorphism, and either the 3′

end of said first probe or the 5′

end of said second probe of said second pair of sensor probes is complementary to a different allele of said single nucleotide polymorphism.

25. The method of claim 23, wherein said detector oliognucleotide further comprises a microsphere, said microsphere comprising said detectable label for said detector oligonucleotide.

26. The method of claim 25, wherein said microsphere is a Luminex microsphere.

27. The method of claim 23, wherein said labels are detected by flow cytometry.

28. The method of claim 23, wherein said determination of the hybridization of said detector oligonucleotides to said amplified ligated sensor probes is quantitative.

29. The method of claim 23, wherein said ligation is achieved using a T4 ligase.

30. The method of claim 23, wherein said at least one target polynucleotide is obtained from a plant or an animal.

31. The method of claim 23, wherein said at least one target polynucleotide comprises DNA

32. The method of claim 23, wherein said at least one target polynucleotide comprises RNA.

33. The method of claim 23, wherein said detector oligonucleotide hybridizes to a portion of said ligated sensor probes that comprises said single nucleotide polymorphism.

34. The method of claim 23, wherein said amplification uses primers that bind to said common primer binding sites.

35. The method of claim 34, wherein said amplification is by the polymerase chain reaction.

36. The method of claim 23, wherein said population of polynucleotides comprises at least 20 different target polynucleotides.

37. The method of claim 23, wherein said population of polynucleotides comprises at least 50 different target polynucleotides.

38. The method of claim 23, wherein said population of polynucleotides comprises at least 100 different target polynucleotides.

39. The method of claim 23, wherein said portion of each sensor probe in a sensor probe pair that is complementary to the target polynucleotide is from about 15 nucleotides long to about 30 nucleotides long.

40. The method of claim 39, wherein said portion of each sensor probe in a sensor probe pair that is complementary to the target polynucleotide is about 20 nucleotides long.

41. The method of claim 23, wherein said common primer binding site is from about 12 nucleotides long to about 24 nucleotides long.

42. The method of claim 41, wherein said common primer site is about 18 nucleotides long.

43. The method of claim 23, wherein said detector oligonucleotide is from about 18 nucleotides long to about 30 nucleotides long.

44. The method of claim 23, wherein each of said sensor probes comprises a first portion complementary to said target polynucleotide and said detector oligonucleotide;

a second portion complementary to said target polynucleotide, but not complementary to said detector oligonucleotide; and

a third portion comprising a common primer binding site that is not complementary to either said target polynucleotide or said detector oligonucleotide.

45. The method of claim 44, wherein when hybridized to said target polynucleotide, said first portions of each of the sensor probes in a sensor probe pair are adjacent to each other.

48. A method of diagnosing a disease, condition, disorder, or predisposition associated with a single nucleotide polymorphism (SNP) comprising:

obtaining a population of polynucletotides from a cell or tissue of a test subject;

determining the presence, absence or frequency of an allele of at least one SNP of interest in said population of polynucleotides using the method of claim 23;

and

comparing the presence, absence or frequency of said allele in the population of polynucleotides from said test subject to the presence, absence or frequency of said allele in a population of polynucleotides obtained from a reference subject known to have said disease, condition, disorder, or predisposition.

49. A method of diagnosing a disease, condition, disorder, or predisposition associated with a single nucleotide polymorphism (SNP) comprising:

obtaining a population of polynucletotides from a cell or tissue of a test subject;

determining the presence, absence or frequency of at least one allele of at least one SNP of interest in said population of polynucleotides using the method of claim 24;

and

comparing the presence, absence or frequency of said allele in the population of polynucleotides from said test subject to the presence, absence or frequency of said allele in a population of polynucleotides obtained from a reference subject known to have said disease, condition, disorder, or predisposition.

52. A kit, comprising at least one pair of sensor probes for at least one target polynucleotide, at least one detector oligonucleotide for each pair of sensor probes, and instructions for carrying out the method of claim 23.

53. The kit of claim 52, wherein said at least one detector oligonucleotide further comprises a labeled microbead or microsphere.

54. A kit, comprising at least one pair of sensor probes for at least one target polynucleotide, at least one detector oligonucleotide for each pair of sensor probes, and instructions for carrying out the method of claim 24.

55. The kit of claim 54, wherein said at least one detector oligonucleotide further comprises a labeled microbead or microsphere.