Multiplexed sequential ligation-based detection of genetic variants

• US 9,206,417 B2
• Filed: 07/19/2013
• Issued: 12/08/2015
• Est. Priority Date: 07/19/2012
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Ariosa Diagnostics Incorporated (Roche Holding AG)
• Inventors: Zahn, Jacob, Oliphant, Arnold, Mohseni, Morassa
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/946,392
• Publication Number:
  US 20140024538A1
• Time in Patent Office: 872 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

The present invention provides multiplexed sequential ligation-based analysis of genetic variants in a mixed sample, including copy number variations and single nucleotide polymorphisms. The invention employs the techniques of sequential ligation and amplification.

Claims

1. A method for identifying a genomic region of interest from a single source in a sample comprising DNA from two different sources, comprising the steps of:

providing a sample comprising DNA from two different sources;

introducing to the sample a first set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in a genomic region of interest, a second fixed sequence oligonucleotide complementary to a 5′

region in the genomic region of interest, and one or more bridging oligonucleotides that hybridize to the genomic region of interest between and adjacent to the first and second fixed sequence oligonucleotides of the first set of oligonucleotide probes;

hybridizing the first set of oligonucleotide probes to the genomic region of interest in the sample;

ligating the hybridized oligonucleotides of the first set of oligonucleotide probes to create first ligation products complementary to the genomic region of interest;

introducing to the first ligation products a second set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in the first ligation product, a second fixed sequence oligonucleotide complementary to a 5′

region in the first ligation product, and one or more bridging oligonucleotides that hybridize to the first ligation product between and adjacent to the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes;

hybridizing the second set of oligonucleotide probes to the first ligation products;

ligating the hybridized oligonucleotides of the second set to create second ligation products complementary to the first ligation products;

amplifying the second ligation products to create amplification products; and

analyzing the amplification products, wherein analysis of the amplification products identifies the genomic region of interest from the single source in the sample.

2. The method of claim 1, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a complementary region that overlaps a ligation junction of the first ligation products.

3. The method of claim 1, wherein both the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a complementary region that overlaps a ligation junction of the first ligation products.

4. The method of claim 1, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a region that is complementary to the genomic region of interest.

5. The method of claim 1, wherein both fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a region that is complementary to the genomic region of interest.

6. The method of claim 1, further comprising a step of amplifying the first ligation products after the first ligating step and before the second introducing step.

7. The method of claim 6, wherein the amplification is linear.

8. The method of claim 6, wherein the amplification is exponential.

9. The method of claim 1, wherein the method is performed for two or more genomic regions of interest from the single source.

10. The method of claim 9, wherein at least 24 different regions of interest are interrogated.

11. The method of claim 10, wherein at least 46 different regions of interest are interrogated.

12. The method of claim 11, wherein at least 92 different regions of interest are interrogated.

13. The method of claim 1, wherein the first and/or second sets of oligonucleotide probes comprise more than one bridging oligonucleotide.

14. The method of claim 1, wherein the first and second sets of oligonucleotide probes comprise a single bridging oligonucleotide.

15. The method of claim 1, wherein the amplification of the second ligation products is linear.

16. The method of claim 1, wherein the amplification of the second ligation products is exponential.

17. A method for identifying a genomic region of interest from a single source in a sample comprising DNA from two different sources, comprising the steps of:

providing a sample comprising DNA from two different sources;

introducing to the sample a first set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in a genomic region of interest and a second fixed sequence oligonucleotide complementary to a 5′

region in the genomic region of interest, wherein the first and second fixed sequence oligonucleotides are complementary to non-adjacent regions in the genomic region of interest;

hybridizing the first set of oligonucleotide probes to the genomic region of interest in the sample;

extending the region between the first fixed sequence oligonucleotide and the second fixed sequence oligonucleotide of the first set of oligonucleotide probes with a polymerase and dNTPs to create contiguously hybridized oligonucleotides of the first set of oligonucleotide probes complementary to the genomic region of interest;

ligating the contiguously hybridized oligonucleotides of the first set to of oligonucleotide probes create first ligation products complementary to the genomic region of interest;

introducing to the first ligation products a second set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in the first ligation product and a second fixed sequence oligonucleotide complementary to a 5′

region in the first ligation product, wherein the first and second fixed sequence oligonucleotides are complementary to non-adjacent regions in the first ligation product;

hybridizing the second set of oligonucleotide probes to the first ligation products;

extending the region between the first fixed sequence oligonucleotide and the second fixed sequence oligonucleotide of the second set with a polymerase and dNTPs to create contiguously hybridized oligonucleotides of the second set of oligonucleotide probes complementary to the first ligation product;

ligating the contiguously hybridized oligonucleotides of the second set of oligonucleotide probes to create second ligation products complementary to the first ligation products;

amplifying the second ligation products to create amplification products; and

analyzing the amplification products, wherein the analysis of the amplification products identifies the genomic region of interest from the single source in the sample.

18. The method of claim 17, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a complementary region that overlaps a ligation junction of the first ligation products.

19. The method of claim 17, wherein both the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a complementary region that overlaps with a ligation junction of the first ligation products.

20. The method of claim 17, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a region that is complementary to the genomic region of interest.

21. The method of claim 17, wherein both fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a region that is complementary to the genomic region of interest.

22. The method of claim 17, further comprising a step of amplifying the first ligation products after the first ligating step and before the second introducing step.

23. The method of claim 22, wherein the amplification is linear.

24. The method of claim 22, wherein the amplification is exponential.

25. The method of claim 17, wherein the method is performed for two or more genomic regions of interest from the single source.

26. The method of claim 25, wherein at least 24 different regions of interest are interrogated.

27. The method of claim 26, wherein at least 46 different regions of interest are interrogated.

28. The method of claim 27, wherein at least 92 different regions of interest are interrogated.

29. The method of claim 28, wherein at least 180 different regions of interest are interrogated.

30. The method of claim 29, wherein at least 360 different regions of interest are interrogated.

31. The method of claim 17, wherein the amplification of the second ligation products is linear.

32. The method of claim 17, wherein the amplification of the second ligation products is exponential.

33. A method for identifying a genomic region of interest from a single source in a sample comprising DNA from two different sources, comprising the steps of:

providing a sample comprising DNA from two different sources;

introducing to the sample a first set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in a genomic region of interest and a second fixed sequence oligonucleotide complementary to a 5′

region in the genomic region of interest;

hybridizing the first set of oligonucleotide probes to the genomic region of interest in the sample;

ligating the hybridized oligonucleotides of the first set of oligonucleotide probes to create first ligation products complementary to the genomic region of interest;

introducing to the first ligation products a second set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in the first ligation product and a second fixed sequence oligonucleotide complementary to a 5′

region in the first ligation product;

hybridizing the second set of oligonucleotide probes to the first ligation products;

ligating the hybridized oligonucleotides of the second set of oligonucleotide probes to create second ligation products complementary to the first ligation products;

amplifying the second ligation products to create amplification products; and

analyzing the amplification products, wherein the analysis of the amplification products identifies the genomic region of interest from the single source in the sample.

34. The method of claim 33, wherein the first set of oligonucleotide probes further comprises one or more bridging oligonucleotides that hybridize to the genomic region of interest between and adjacent to the first and second fixed sequence oligonucleotides of the first set of oligonucleotide probes.

35. The method of claim 33, wherein the second set further comprises one or more bridging oligonucleotides that hybridize to the first ligation products between and adjacent to the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes.

36. The method of claim 33, wherein the oligonucleotides of the first set of oligonucleotide probes are complementary to non-adjacent regions in the genomic region of interest, and wherein the region between the first fixed sequence oligonucleotide and the second fixed sequence oligonucleotide of the first set is extended with a polymerase and dNTPs to create contiguously complementary oligonucleotides of the first set.

37. The method of claim 33, wherein the oligonucleotides of the second set of oligonucleotide probes are complementary to non-adjacent regions in the first ligation products, and wherein the region between the first fixed sequence oligonucleotide and the second fixed sequence oligonucleotide of the second set is extended with a polymerase and dNTPs to create contiguously complementary oligonucleotides of the second set.

38. The method of claim 33, wherein the fixed sequence oligonucleotides of the first set of oligonucleotide probes are complementary to adjacent regions in the genomic region of interest.

39. The method of claim 33, wherein the fixed sequence oligonucleotides of the second set of oligonucleotide probes are complementary to adjacent regions in the first ligation products.

40. The method of claim 33, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a complementary region that overlaps a ligation junction of the first ligation products.

41. The method of claim 33, wherein both the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a complementary region that overlaps with a ligation junction of the first ligation products.

42. The method of claim 33, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises the region of a fixed sequence oligonucleotide of the first set of oligonucleotide probes that is complementary to the genomic region of interest.

43. The method of claim 33, wherein both fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a region of the fixed sequence oligonucleotides that is complementary to the genomic region of interest.

44. The method of claim 33, further comprising a step of amplifying the first ligation products after the first ligating step and before the second introducing step.

45. The method of claim 44, wherein the amplification is linear.

46. The method of claim 44, wherein the amplification is exponential.

47. The method of claim 33, wherein the method is performed for two or more genomic regions of interest from the single source.

48. The method of claim 47, wherein at least 24 different regions of interest are interrogated.

49. The method of claim 48, wherein at least 46 different regions of interest are interrogated.

50. The method of claim 49, wherein at least 92 different regions of interest are interrogated.

51. The method of claim 33, wherein the first and/or second sets of oligonucleotide probes comprise more than one bridging oligonucleotide.

52. The method of claim 33, wherein the first and second sets of oligonucleotide probes comprise a single bridging oligonucleotide.

53. The method of claim 33, wherein the amplification of the second ligation products is linear.

54. The method of claim 33, wherein the amplification of the second ligation products is exponential.