Nucleic acid size detection method

• US 20080124709A1
• Filed: 10/05/2006
• Published: 05/29/2008
• Est. Priority Date: 10/05/2006
• Status: Active Grant

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated), U.S. Genomics, Inc (Toxic Reports LLC)
• Original Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Huang, Donghui, Strom, Charles M., Potts, Steven J., Rooke, Jenny Ellen
• Granted Patent: US 8,163,480 B2
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: Y10T 436/17 Nitrogen containing

Abstract

The present invention provides methods of determining the size of a particular nucleic acid segment of interest in a sample of nucleic acids through fragmentation of DNA, size fractionation, an optional second fragmentation, and identification using a marker sequence. In particular aspects, an expansion or reduction of tandem repeat sequences can be detected. In further aspects, carriers and individuals afflicted with fragile X syndrome or other diseases associated with tandem repeats can be distinguished from normal individuals.

Claims

1. A method for determining the size of a nucleic acid segment in a nucleic acid sample, comprising,

fragmenting the nucleic acid in the sample into fragments, wherein said segment is associated with a marker sequence in said fragment,

separating said fragments into fractions according to size under conditions in which the fragment(s) containing the segment will be located in said fractions according to the size of the segment, and

identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the size of the segment is determined by the fraction in which it is identified.

2. A method according to claim 1, wherein said fragments are prepared using one or more restriction endonucleases.

3. A method according to claim 1, wherein said fractions are chosen to separate fragments containing segments having a normal size from fragments containing segments having an abnormal size.

4. A method according to claim 1, wherein said fragments are size-separated into a number of fractions selected from the group consisting of 2-16.

5. A method according to claim 1, wherein said fractions of size-separated nucleic acids are obtained using electrophoresis.

6. A method according to claim 5, wherein said electrophoresis is capillary electrophoresis.

7. A method according to claim 1, wherein said fractions of size-separated nucleic acid fragments are obtained by chromatography.

8. A method according to claim 1, wherein after size-separation of the fragments all or a portion of the segment is cleaved from said marker sequence.

9. A method according to claim 8, wherein said segment is cleaved using a restriction endonuclease.

10. A method according to claim 1, wherein said detecting further comprises amplifying said marker sequence.

11. A method according to claim 10, wherein said amplifying is accomplished with polymerase chain reaction (PCR).

12. A method according to claim 11, wherein said PCR includes a labeled primer.

13. The method of claim 1, wherein said detecting is accomplished using the Taqman PCR detection system.

14. A method according to claim 1, wherein said marker sequence is detected by hybridizing to two probes that hybridize simultaneously to the marker sequence.

15. A method according to claim 1, wherein said segment comprises a tandem repeat region.

16. A method according to claim 15, wherein the size determined is the number of repeats in said tandem repeat region.

18. A method according to claim 15, wherein said tandem repeats are trinucleotide tandem repeats.

19. A method according to claim 15, wherein said tandem repeat is associated with a gene selected from the group consisting of DRPLA, EPM1, FRAXA, FMR1, FRDA, huntingtin, JPH-3, AR, DM1, DM2, SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, PABPN1 and HOXD13.

20. A method according to claim 19 wherein said tandem repeat is associated with a gene selected from the group consisting of FMR1, FRDA, and DM1.

21. A method according to claim 20, wherein said gene is FMR1.

60. A method of determining the size of a tandem repeat segment in a sample of nucleic acids, said method comprising

a) measuring the size of a tandem repeat segment by a direct method which comprises amplification of the tandem repeat segment,

b) measuring the size by the method of claim 1, and

c) using the information obtained in steps a) and b) to determine the size of the tandem repeat region.

61. A method according to claim 60, wherein the amplification of step a) is accomplished with polymerase chain reaction (PCR).

62. A method according to claim 61, further comprising sizing of the amplified tandem repeat region using electrophoresis.

17. A method according to claim 17, wherein said fractions are chosen to separate fragments with a normal number of tandem repeats from fragments containing an abnormal number of tandem repeats.

22. A method of detecting a mutation in a tandem repeat segment of a gene in a genomic nucleic acid sample, wherein said mutation is characterized by a change in the number of repeats compared to the number of repeats in the wild type allele, said method comprising

fragmenting the nucleic acid in the sample into fragments, wherein said tandem repeat segment is associated with a marker sequence in said fragment;

separating said fragments into fractions according to size under conditions in which the fragment(s) containing the tandem repeat segment will be located in said fractions according to the number of repeats in the tandem repeat segment;

identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, and

comparing the number of repeats in the tandem repeat segment from the nucleic acid sample to the number in the corresponding wild type allele, wherein a number of repeats differing from the number in the wild type allele is indicative of a mutation.

23. A method according to claim 22, further comprising,

determining if a mutation is a premutation or a full mutation by comparing the number of repeats in the tandem repeat segment from the nucleic acid sample to the number in the corresponding full mutation allele, wherein a number of repeats greater than the wild type allele but less than the full mutation is indicative of a premutation allele, whereas a number of repeats greater than or equal to the full mutation is indicative of a full mutation allele.

24. A method according to claim 22, wherein said fragments are obtained using one or more restriction endonucleases.

25. A method according to claim 24, wherein said one or more restriction endonucleases are selected from the group consisting of AluI, SphI, BmtI, BlpI, MlyI, and BstNI.

26. A method according to claim 25, wherein said one or more restriction endonuclease is AluI.

27. A method according to claim 25, wherein said one or more restriction endonucleases are SphI and BmtI.

28. A method according to claim 25, wherein said one or more restriction endonucleases are BlpI and MylI.

29. A method according to claim 22, wherein said fractions of size separated nucleic acids are obtained using electrophoresis.

30. A method according to claim 31, wherein said electrophoresis is capillary electrophresis.

31. A method according to claim 22, wherein said fractions of size separated nucleic acids are obtained using chromatography.

32. A method according to claim 22, wherein said fractions are chosen to separate fragments containing tandem repeat segments having a normal number of tandem repeats from fragments containing tandem repeat segments having an abnormal number of repeats.

33. A method according to claim 22, wherein said nucleic acid fragments are size-separated into a number of fractions selected from the group consisting of 2-16.

34. A method according to claim 22, wherein after size-separation of the fragments all or a portion of the particular nucleic acid segment is cleaved from said marker sequence.

35. A method according to claim 34, wherein the particular nucleic acid segment is cleaved using a restriction endonuclease.

36. A method according to claim 35, wherein said restriction endonuclease is selected from the group consisting of BsaWI, HphI, BbvI, BstNI, Hpy1881, SmlI, and BmtI.

37. A method according to claim 22, wherein said detecting further comprises amplifying said marker.

38. A method according to claim 37, wherein said amplifying is accomplished with polymerase chain reaction (PCR).

39. A method according to claim 38 wherein said amplifying employs a detectably labeled primer.

40. A method according to claim 39 said detecting is accomplished with electrophoresis.

41. The method of claim 22, wherein said detecting is accomplished using the Taqman PCR detection system.

42. A method according to claim 22, wherein said marker sequence is detected by hybridizing to two probes that hybridize simultaneously to the marker sequence.

43. A method according to claim 22, wherein said tandem repeats are trinucleotide tandem repeats.

44. A method of distinguishing between normal individuals and that are normal, those that are carriers of fragile X syndrome, and those that are afflicted with fragile X syndrome, said method comprising,

fragmenting the nucleic acid in the sample from the individual into fragments, wherein said tandem repeat segment of the FMR1 gene is associated with a marker sequence in said fragment,

separating said fragments into fractions according to size under conditions in which the fragment(s) containing the tandem repeat segment having a normal number of repeats will be located in a first fraction;

fragments containing a tandem repeat segment having a premutation will be located in a second fraction; and

fragments having a tandem repeat region having a full mutation will be located in a third fraction,

identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, wherein

a positive result in only the first fraction indicates the individual is normal;

a positive result in the second fraction indicates the individual is a carrier; and

a positive result in the third fraction indicates the individual is afflicted with fragile X syndrome.

45. A method according to claim 44, wherein said fragments are obtained using one or more restriction endonucleases.

46. A method according to claim 45, wherein said one or more restriction endonucleases are selected from the group consisting of AluI, SphI, BmtI, BlpI, MlyI, and BstNI.

47. A method according to claim 46, wherein said one or more restriction endonuclease is AluI.

48. A method according to claim 46, wherein said one or more restriction endonucleases are SphI and BmtI.

49. A method according to claim 46, wherein said one or more restriction endonucleases are BlpI and MylI.

50. A method according to claim 44, wherein said fractions of size separated nucleic acids are obtained using electrophoresis.

51. A method according to claim 44, wherein said fractions of size separated nucleic acids are obtained using chromatography.

52. A method according to claim 44, wherein after size-separation of the fragments all or a portion of the particular nucleic acid segment is cleaved from said marker sequence.

53. A method according to claim 52, wherein the particular nucleic acid segment is cleaved using a restriction endonuclease.

54. A method according to claim 53, wherein said restriction endonuclease is selected from the group consisting of BsaWI, HphI, BbvI, BstNI, Hpy1881, SmlI, and BmtI.

55. A method according to claim 44, wherein said detecting further comprises amplifying said marker.

56. A method according to claim 55, wherein said amplifying is accomplished with polymerase chain reaction (PCR).

57. A method according to claim 56 wherein said amplifying employs a detectably labeled primer.

58. A method according to claim 57 said detecting is accomplished with electrophoresis.

59. The method of claim 44, wherein said detecting is accomplished using the Taqman PCR detection system.

63. A kit for detecting the size of a particular nucleic acid segment in a sample comprising a primer pair for amplifying a marker nucleotide sequence upstream or downstream of the particular nucleic acid segment, and one or more restriction endonucleases for cleaving the nucleic acid sample to generate a fragment of the nucleic acid sample which contains the particular nucleic acid segment and the upstream or downstream marker sequence, wherein said particular nucleic acid segment is a tandem repeat sequence.

64. A kit according to claim 63, further comprising one or more restriction enzymes for separating the particular nucleic acid segment from the marker sequence.

65. A kit according to claim 63, further comprising one or more controls that verify proper size separation of nucleic acids digested with the restriction endonuclease.

66. A kit according to claim 63, wherein the tandem repeat segment is from the FMR1 gene.