Methods for rapid forensic analysis of mitochondrial DNA

• US 8,407,010 B2
• Filed: 03/17/2008
• Issued: 03/26/2013
• Est. Priority Date: 05/25/2004
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories), Florida Turbine Technologies, Inc. (Kratos Defense & Security Solutions Incorporated)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Hofstadler, Steven A., Hall, Thomas A., Ecker, David J., Blyn, Lawrence B., Eshoo, Mark W., Samant, Vivek, White, Neill
• Primary Examiner(s): ZEMAN, MARY K
• Application Number:
  US12/049,949
• Publication Number:
  US 20090125245A1
• Time in Patent Office: 1,835 Days
• Field of Search: None
• US Class Current: 702/19
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/623 combined with mass spectrometry
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: G16B 50/30 Data warehousing; Computing architectures

Abstract

The present invention provides methods for rapid forensic analysis of mitochondrial DNA by amplification of a segment of mitochondrial DNA containing restriction sites, digesting the mitochondrial DNA segments with restriction enzymes, determining the molecular masses of the restriction fragments and comparing the molecular masses with the molecular masses of theoretical restriction digests of known mitochondrial DNA sequences stored in a database.

Claims

1. A method of forensic analysis of a sample comprising mitochondrial DNA comprising:

selecting a region of mitochondrial DNA comprising at least one restriction site whereat a restriction enzyme cleaves said mitochondrial DNA to produce a plurality of restriction fragments;

populating a relational database of known mitochondrial DNA sequences with base compositions which correspond to theoretical restriction fragments obtained from theoretical digestion of each member of said database at said at least one restriction site;

selecting a primer pair with which to amplify said region of mitochondrial DNA in said sample;

amplifying said region of mitochondrial DNA in said sample to produce an amplification product;

digesting said amplification product with at least one restriction enzyme to produce a plurality of restriction fragments;

experimentally determining the base compositions of each member of said plurality of restriction fragments, wherein said base composition is determined without sequencing said restriction fragments; and

comparing said experimentally determined base compositions with the base compositions of said theoretical digestion of each member of said database, wherein at least one match or lack of a match provides a forensic conclusion.

2. The method of claim 1 wherein said region of mitochondrial DNA comprises HV1.

3. The method of claim 2 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

12;

13, 12;

14, 12;

15, 16;

17, 42;

43, 42;

46, 67;

68, 69;

70, 12;

68, 12;

70, 67;

15, 71;

70, 69;

15 and 69;

68.

4. The method of claim 2 wherein said at least one restriction enzyme is RsaI.

5. The method of claim 1 wherein said region of mitochondrial DNA comprises HV2.

6. The method of claim 5 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

8;

9, 10;

11, 16;

17 and 65;

66.

7. The method of claim 5 wherein said at least one restriction enzyme is HaeIII, HpaII, MfeI, or SspI.

8. The method of claim 5 wherein said at least one restriction enzyme is HpaII, HpyCH4IV, PacI, or EaeI.

9. The method of claim 1 wherein said region of mitochondrial DNA comprises region R1.

10. The method of claim 9 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

18;

19 and 18;

49.

11. The method of claim 9 wherein said at least one restriction enzyme is DdeI, MseI, HaeIII, or MboI.

12. The method of claim 1 wherein said region of mitochondrial DNA comprises region R2.

13. The method of claim 12 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

20;

21 and 50;

51.

14. The method of claim 12 wherein said at least one restriction enzyme is DdeI, HaeIII, MboI, or MseI.

15. The method of claim 1 wherein said region of mitochondrial DNA comprises region R3.

16. The method of claim 15 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

22;

23 and 22;

52.

17. The method of claim 15 wherein said at least one restriction enzyme is DdeI, MseI, MboI, or BanI.

18. The method of claim 1 wherein said region of mitochondrial DNA comprises region R4.

19. The method of claim 18 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

24;

25 and 53;

54.

20. The method of claim 18 wherein said at least one restriction enzyme is DdeI, HpyCH4IV, MseI, or HaeIII.

21. The method of claim 1 wherein said region of mitochondrial DNA comprises region R5.

22. The method of claim 21 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

26;

27 and 55;

56.

23. The method of claim 21 wherein said at least one restriction enzyme is AluI, BfaI, or MseI.

24. The method of claim 1 wherein said region of mitochondrial DNA comprises region R6.

25. The method of claim 24 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

28;

29 and 57;

29.

26. The method of claim 24 wherein said at least one restriction enzyme is DdeI, HaeIII, MboI, MseI, or RsaI.

27. The method of claim 1 wherein said region of mitochondrial DNA comprises region R7.

28. The method of claim 27 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

30;

31 and 58;

31.

29. The method of claim 27 wherein said at least one restriction enzyme is DdeI, HpaII, HaeIII, or MseI.

30. The method of claim 1 wherein said region of mitochondrial DNA comprises region R8.

31. The method of claim 30 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of any one of the following primer pair sequences:

SEQ ID NOs;

32;

33 and 59;

60.

32. The method of claim 30 wherein said at least one restriction enzyme is BfaI, DdeI, EcoRI, or MboI.

33. The method of claim 1 wherein said region of mitochondrial DNA comprises region R9.

34. The method of claim 33 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of the following primer pair sequences:

SEQ ID NOs;

34;

35.

35. The method of claim 33 wherein said at least one restriction enzyme is BfaI, DdeI, HpaII, HpyCH4IV, or MboI.

36. The method of claim 1 wherein said region of mitochondrial DNA comprises region R10.

37. The method of claim 36 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of the following primer pair sequences:

SEQ ID NOs;

34;

35.

38. The method of claim 36 wherein said at least one restriction enzyme is BfaI, HpaII, or MboI.

39. The method of claim 1 wherein said region of mitochondrial DNA comprises region R10.

40. The method of claim 39 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of the following primer pair sequences:

SEQ ID NOs;

36;

37 and 61;

62.

41. The method of claim 39 wherein said at least one restriction enzyme is BfaI, HpaII, or MboI.

42. The method of claim 1 wherein said region of mitochondrial DNA comprises region R11.

43. The method of claim 42 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of the following primer pair sequences:

SEQ ID NOs;

38;

39 and 63;

39.

44. The method of claim 42 wherein said at least one restriction enzyme is BfaI, DdeI, HpyCH4V, or MboI.

45. The method of claim 1 wherein said region of mitochondrial DNA comprises region R12.

46. The method of claim 45 wherein each member of said primer pair has at least 70% sequence identity with the sequence of the corresponding member of the following primer pair sequences:

SEQ ID NOs;

40;

41 and 40;

64.

47. The method of claim 45 wherein said at least one restriction enzyme is BfaI, DdeI, or MseI.

48. The method of claim 1 wherein said base compositions of said restriction fragments are determined from molecular masses.

49. The method of claim 1 wherein said amplifying step comprises polymerase chain reaction.

50. The method of claim 49 wherein said polymerase chain reaction is catalyzed by a polymerase enzyme whose function is modified relative to a native polymerase.

51. The method of claim 50 wherein said modified polymerase enzyme is exo(−

) Pfu polymerase.

52. The method of claim 50 wherein said modified polymerase catalyzes the addition of nucleotide residues to staggered restriction digest products to convert said staggered digest products to blunt-ended digest products.

53. The method of claim 1 wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.

54. The method of claim 1 wherein said database is a human mtDNA population database.

55. The method of claim 48 wherein said molecular masses are determined by ESI-FTICR mass spectrometry.

56. The method of claim 48 wherein said molecular masses are determined by ESI-TOF mass spectrometry.

57. The method of claim 1 further comprising repeating all steps of the method for at least one additional region of mitochondrial DNA.

58. The method of claim 57 wherein said at least one additional region is from HV1, HV2, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12.

59. The method of claim 1 wherein said mitochondrial DNA is human mitochondrial DNA.

60. The method of claim 1 wherein said mitochondrial DNA is animal mitochondrial DNA.

61. The method of claim 1 wherein said mitochondrial DNA is fungal, parasitic, or protozoan DNA.

62. The method of claim 1 wherein said amplified DNA is digested directly without purification.

63. The method of claim 1 wherein said sample of mitochondrial DNA is obtained from saliva, hair, blood, or nail.

64. The method of claim 1 wherein said plurality of restriction fragments are up to about 150 base pairs in length.

65. The method of claim 1, wherein said forensic conclusion comprises the identity of a source of said sample.

66. The method of claim 48, wherein said forensic conclusion comprises the identity of a source of said sample.