Enzymatic ligation-based identification of nucleotide sequences

• US 20030170695A1
• Filed: 12/27/2002
• Published: 09/11/2003
• 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:
  US10/330,774
• Publication Number:
  US 20030170695A1
• 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, detection of single nucleotide polymorphisms, detection of pathogens, and detection of genetically modified cells and organisms. The method can be practiced using purified preparations of nucleic acids or unpurified cell lysates. Also provided are diagnostic methods and kits for conducting the disclosed methods.

Claims

1. A method for detecting a genetically modified cell or organism in a population comprising:

obtaining polynucleotides from at least one sample from said population;

providing a pair of specific sensor probes for at least one transgene contained in a genetically modified cell or organism of interest, 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 transgene and a 5′

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

portion complementary to said transgene and a 3′

portion containing a primer binding site, wherein said complementary portions of said first and second probe are complementary to immediately adjacent regions on said transgene;

combining said polynucleotides with said specific sensor probes under stringent hybridization conditions;

allowing said sensor probes to hybridize to said at least one transgene;

ligating hybridized members of a sensor probe pairs to form ligated sensor probes comprising a ligation site;

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

for each different ligated sensor probe 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 transgene and contains said ligation site;

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 transgene by the identity of the detector oligonucleotide.

2. The method of claim 1, wherein said polynucleotides are contained in an unpurified cell lysate.

3. The method of claim 1, wherein said polynucleotides are contained in a partially purified cell lysate.

4. The method of claim 1, wherein said polynucleotides are DNA, cDNA, RNA, mRNA, polyA RNA, or a mixture thereof.

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

6. The method of claim 5, wherein said detectable label of said microsphere comprises at least two different fluorochromes.

7. The method of claim 1, wherein said primer binding sites of said sensor probe pair are not complementary to said transgene.

8. The method of claim 1, wherein said primer binding site of said first probe of each sensor probe pair comprises a common primer binding site and said primer binding site of said second probe of each sensor probe pair comprises a common primer binding site.

9. The method of claim 1,wherein each of said sensor probes comprises a first portion complementary to said transgene and said detector oligonucleotide;

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

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

10. The method of claim 9, wherein when hybridized to said transgene said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

11. The method of claim 8,wherein each of said sensor probes comprises a first portion complementary to said transgene and said detector oligonucleotide;

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

a third portion comprising said common primer binding site wherein the common primer binding site is not complementary to either said transgene or said detector oligonucleotide.

12. The method of claim 11, wherein when hybridized to said transgene said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

13. The method of claim 1, wherein said genetically modified cell or organism is a plant, an animal, a bacteria, a yeast, a fungus or a virus.

14. The method of claim 1, wherein said detector oligonucleotides are from about 15 nucleotides long to about 30 nucleotides long.

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

16. The method of claim 1, wherein said detectable labels are quantitative labels.

17. The method of claim 1, wherein said detectable labels are detected by flow cytometry.

18. A method for detecting a pathogen in a subject or composition comprising:

obtaining polynucleotides from at least one sample from said subject or composition;

providing a pair of specific sensor probes for at least one target polynucleotide sequence characteristic of said pathogen, 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 sequence and a 5′

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

portion complementary to said target polynucleotide sequence and a 3′

portion containing a primer binding site, wherein said complementary portions of said first and second probe are complementary to immediately adjacent regions on said target polynucleotide sequence;

combining said polynucleotides with said specific sensor probes under stringent hybridization conditions;

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

ligating hybridized members of a sensor probe pairs to form ligated sensor probes comprising a ligation site;

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

for each different ligated sensor probe 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 sequence and contains said ligation site;

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 sequence by the identity of the detector oligonucleotide, said polynucleotide sequence used to identify said pathogen.

19. The method of claim 18, wherein said polynucleotides are contained in an unpurified cell lysate.

20. The method of claim 18, wherein said polynucleotides are contained in a partially purified cell lysate.

21. The method of claim 18, wherein said primer binding sites of said sensor probe pair are not complementary to said target polynucleotide sequence.

22. The method of claim 18, wherein said primer binding site of said first probe of each sensor probe pair comprises a common primer binding site and said primer binding site of said second probe of each sensor probe pair comprises a common primer binding site.

23. The method of claim 18,wherein each of said sensor probes comprises a first portion complementary to said target polynucleotide sequence and said detector oligonucleotide;

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

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

24. The method of claim 23, wherein when hybridized to said target polynucleotide sequence said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

25. The method of claim 22,wherein each of said sensor probes comprises a first portion complementary to said target polynucleotide sequence and said detector oligonucleotide;

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

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

26. The method of claim 25, wherein when hybridized to said target polynucleotide sequence said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

27. The method of claim 18, wherein said polynucleotides are DNA, cDNA, RNA, mRNA, polyA RNA or a mixture thereof.

28. The method of claim 18, wherein said detector oligonucleotide further comprises a microsphere, said microsphere comprising said detectable label for said detector oligonucleotide.

29. The method of claim 28, wherein said detectable label of said microsphere comprises at least two different fluorochromes.

30. The method of claim 18, wherein said detector oligonucleotides are from about 15 nucleotides long to about 30 nucleotides long.

31. The method of claim 18, wherein said primer binding site is from about 12 nucleotides long to about 24 nucleotides long.

32. The method of claim 18, wherein said detectable labels are quantitative labels.

33. The method of claim 18, wherein said detectable labels are detected by flow cytometry.

34. The method of claim 18, wherein said pathogen is a bacteria, a virus, a yeast, a fungus, or a parasite.

35. The method of claim 18, wherein said subject is an animal or a plant.

36. The method of claim 18, wherein said composition is a feedstuff, a foodstuff, a biological fluid, a tissue, a biopsy, a culture medium, or a pharmaceutical composition.

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

obtaining from a subject a population of cells, wherein said population comprises cells containing at least one target polynucleotide containing a single nucleotide polymorphism;

lysing said cells to form a cell lysate containing said target polynucleotide and wherein said lysate is not further purified;

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 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 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 ligated sensor probes comprising a ligation site 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 different ligated sensor probe providing at least one class of detector oligonucleotide, said oligonucleotides 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 and which contains said ligation site;

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.

38. The method of claim 37 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.

39. The method of claim 37, wherein said primer binding site of said first probe of each sensor probe pair comprises a common primer binding site and said primer binding site of said second probe of each sensor probe pair comprises a common primer binding site.

40. The method of claim 37,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 to said detector oligonucleotide; and

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

41. The method of claim 40, wherein when hybridized to said transgene said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

42. The method of claim 39,wherein each of said sensor probes comprises a first portion complementary to said transgene and said detector oligonucleotide;

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

a third portion comprising said common primer binding site wherein the common primer binding site is not complementary to either said transgene or said detector oligonucleotide.

43. The method of claim 42, wherein when hybridized to said transgene said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

44. The method of claim 37, wherein said detector oligonucleotide further comprises a microsphere, said microsphere comprising said detectable label for said detector oligonucleotide.

45. The method of claim 44, wherein said microsphere comprises two different fluorochromes.

46. The method of claim 37, wherein said labels are detected by flow cytometry.

47. The method of claim 37, wherein said determination of the hybridization of said detector oligonucleotides to said amplified ligated sensor probes is quantitative.

48. The method of claim 37, wherein said at least one target polynucleotide is obtained from a plant or an animal.

49. The method of claim 37, wherein said at least one target polynucleotide comprises DNA, cDNA, RNA, mRNA, poly A RNA, or a mixture thereof.

50. The method of claim 37, wherein said population of polynucleotides comprises at least 20 different target polynucleotides.

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

52. The method of claim 37, wherein said population of polynucleotides comprises at least 100 different target polynucleotides.

53. The method of claim 37, 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.

54. The method of claim 37, wherein said common primer binding site is from about 12 nucleotides long to about 24 nucleotides long.

55. The method of claim 37, wherein said detector oligonucleotide is from about 18 nucleotides long to about 30 nucleotides long.

56. The method of claim 37, wherein said cell lysate is partially purified.

57. A method for determining polynucleotide expression comprising:

obtaining a population of cells, wherein said population comprises cells containing at least one target polynucleotide of interest;

lysing said cells to form a cell lysate containing said target polynucleotide and wherein said lysate is not further purified;

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 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 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 ligated sensor probes containing a ligation site;

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

for each different ligated sensor probe 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 and which contains said ligation site;

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.

58. The method of claim 57, wherein said primer binding site of said first probe of each sensor probe pair comprises a common primer binding site and said primer binding site of said second probe of each sensor probe pair comprises a common primer binding site.

59. The method of claim 57,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 to said detector oligonucleotide; and

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

60. The method of claim 59, wherein when hybridized to said transgene said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

61. The method of claim 58,wherein each of said sensor probes comprises a first portion complementary to said transgene and said detector oligonucleotide;

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

a third portion comprising said common primer binding site wherein the common primer binding site is not complementary to either said transgene or said detector oligonucleotide.

62. The method of claim 61, wherein when hybridized to said transgene said first portions of each of the sensor probes in said sensor probe pair are adjacent to each other.

63. The method of claim 57, wherein said detector oligonucleotide further comprises a microsphere, said microsphere comprising said detectable label for said detector oligonucleotide.

64. The method of claim 63, wherein said microsphere comprises two different fluorochromes.

65. The method of claim 57, wherein said labels are detected by flow cytometry.

66. The method of claim 57, wherein said determination of the hybridization of said detector oligonucleotides to said amplified ligated sensor probes is quantitative.

67. The method of claim 57, wherein said at least one target polynucleotide is obtained from a plant, an animal, a bacteria, a yeast or a fungus.

68. The method of claim 57, wherein said at least one target polynucleotide comprises cDNA, RNA, mRNA, poly A RNA, or a mixture thereof.

69. The method of claim 57, wherein said cells comprises at least 20 different target polynucleotides.

70. The method of claim 57, wherein said cells comprises at least 50 different target polynucleotides.

71. The method of claim 57, wherein said cells comprises at least 100 different target polynucleotides.

72. The method of claim 57, 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.

73. The method of claim 57, wherein said common primer binding site is from about 12 nucleotides long to about 24 nucleotides long.

74. The method of claim 57, wherein said detector oligonucleotide is from about 18 nucleotides long to about 30 nucleotides long.

75. The method of claim 57, wherein said cell lysate is partially purified.

76. A method for determining a change in polynucleotide expression comprising:

obtaining a first population of cells, wherein said population comprises cells containing at least one target polynucleotide of interest;

determining the expression of said at least one polynucleotide of interest in said first population by the method of claim 57;

obtaining a second population of cells, wherein said population comprises cells containing said at least one target polynucleotide of interest;

determining the expression of said at least one polynucleotide of interest in said second population by the method of claim 57;

comparing the expression of said at least one polynucleotide of interest in said first population to the expression of said at least one polynucleotide of interest in said second population.