Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy

• US 7,666,588 B2
• Filed: 09/12/2003
• Issued: 02/23/2010
• Est. Priority Date: 03/02/2001
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: White, Neil, Hofstadler, Steven A., Sampath, Rangarajan, Drader, Jared J., Griffey, Richard H., Hall, Thomas A., McNeil, John, Blyn, Lawrence B., Eshoo, Mark W., Ecker, David J., Crooke, Stanley T., Hannis, James C., Samant, Vivek, Jiang, Yun
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US10/660,998
• Publication Number:
  US 20040161770A1
• Time in Patent Office: 2,356 Days
• Field of Search: 435/5, 435/91.2, 435/6, 536/24.33, 536/24.31, 536/24.32, 536/23.1
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 2333/28 from Vibrionaceae (F)
• CPC Class Codes: G01N 2333/32 from Bacillus (G)
• CPC Class Codes: G01N 2333/35 from Mycobacteriaceae (F)
• CPC Class Codes: G01N 2333/36 from Actinomyces; from Streptomyces (G)

Abstract

The present invention provides methods for rapid forensic analysis of mitochondrial DNA and methods for characterizing heteroplasmy of mitochondrial DNA, which can be used to assess the progression of mitochondrial diseases.

Claims

1. A forensic method of mitochondrial DNA analysis comprising the steps of:

providing a forensic evidence sample;

amplifying two or more segments of mitochondrial DNA obtained from said forensic evidence sample to obtain two or more amplification products;

determining the molecular masses of said two or more amplification products by mass spectrometry, without sequencing said two or more amplification products; and

comparing said molecular masses of said two or more amplification products with at least one database comprising a plurality of known molecular masses from said two or more segments of mitochondrial DNA from a plurality of subjects, thereby reaching a forensic conclusion.

2. The forensic method of claim 1, further comprising digesting said two or more amplification products with one or more restriction enzymes to produce restriction fragments before said mass spectrometry.

3. The forensic method of claim 2, wherein said one or more restriction enzymes are selected from the group consisting of RsaI , HpaII, HpyCH4IV, PacI, and EaeI.

4. The forensic method of claim 2, further comprising determining the molecular masses of said restriction fragments by mass spectrometry, without sequencing said restriction fragments.

5. The forensic method of claim 2, further comprising comparing said molecular masses of said restriction fragments with at least one database comprising a plurality of known molecular masses from said two or more segments of mitochondrial DNA from a plurality of subjects, thereby reaching a forensic conclusion.

6. The forensic method of claim 1, wherein said subjects are animals.

7. The forensic method of claim 6, wherein said animals are humans.

8. The forensic method of claim 1, wherein said subjects are nonhuman eukaryotic organisms, fungi, parasites or protozoa.

9. The forensic method of claim 1, wherein said two or more segments of mitochondrial DNA comprise a portion of a hypervariable region of mitochondrial DNA.

10. The forensic method of claim 9, wherein said hypervariable region comprises at least one of HVR1 or HVR2.

11. The forensic method of claim 1, wherein said two or more amplification products are generated from two or more hypervariable portions of the noncoding region of mitochondrial DNA using flanking primers.

12. The forensic method of claim 1, wherein said two or more segments of mitochondrial DNA comprise the entire mitochondrial DNA of said subject.

13. The forensic method of claim 1, wherein said forensic conclusion comprises identification of at least one subject from whom said forensic evidence sample is obtained by comparing said molecular masses of said two or more amplification products with said plurality of known molecular masses in said at least one database.

14. The forensic method of claim 13, wherein said forensic conclusion is the identification of a criminal.

15. The forensic method of claim 13, wherein said forensic conclusion is the identification of a crime victim.

17. The forensic method of claim 1, wherein said forensic conclusion further comprises determining the movement of at least one subject from whom said forensic evidence sample is obtained by mitochondrial DNA analysis of a plurality of forensic evidence samples obtained from a plurality of locations.

18. The forensic method of claim 1, wherein said mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry.

19. The forensic method of claim 1, wherein said at least one database is a Federal Bureau of Investigation mitochondrial DNA database.

46. The forensic method of claim 1, wherein said forensic conclusion comprises identification of a missing person, detection and identification of a known bioagent, detection and identification of an unknown bioagent, elimination of an individual as a crime suspect, identification of an individual as a crime suspect, identification of a location as a crime scene, identification of a location as an accident scene, identification of evidence useful in a court of law, identification of evidence useful in a criminal investigation, or identification of one or more biological samples from a crime scene.

16. The method of 1, further comprising determining the relative amounts of said one or more amplification products from the abundance of mass spectral peaks corresponding to said one or more amplification products.

20. A forensic method of mitochondrial DNA analysis comprising the steps of:

providing a forensic evidence sample;

amplifying two or more segments of mitochondrial DNA obtained from said forensic evidence sample to obtain two or more amplification products;

determining the molecular masses of said two or more amplification products by mass spectrometry, without sequencing said two or more amplification products;

calculating base compositions of said two or more amplification products from said molecular masses; and

comparing said base compositions of said two or more amplification products with at least one database comprising a plurality of known base compositions from said two or more segments of mitochondrial DNA from a plurality of subjects, thereby reaching a forensic conclusion.

21. The forensic method of claim 20, further comprising digesting said two or more amplification products with one or more restriction enzymes to produce restriction fragments before said mass spectrometry.

22. The forensic method of claim 21, wherein said one or more restriction enzymes are selected from the group consisting of RsaI , HpaII, HpyCH4IV, PacI, and EaeI.

23. The forensic method of claim 21, further comprising determining the base compositions of said restriction fragments by mass spectrometry, without sequencing said restriction fragments.

24. The forensic method of claim 21, further comprising comparing said base compositions of said restriction fragments with at least one database comprising a plurality of known base compositions from said two or more segments of mitochondrial DNA from a plurality of subjects, thereby reaching a forensic conclusion.

25. The forensic method of claim 20, wherein said subjects are animals.

26. The forensic method of claim 25, wherein said animals are humans.

27. The forensic method of claim 20, wherein said subjects are nonhuman eukaryotic organisms, fungi, parasites or protozoa.

28. The forensic method of claim 20, wherein said two or more segments of mitochondrial DNA comprise a portion of a hypervariable region of mitochondrial DNA.

29. The forensic method of claim 28, wherein said hypervariable region comprises at least one of HVR1 or HVR2.

30. The forensic method of claim 20, wherein said two or more amplification products are generated from two hypervariable portions of the noncoding region of mitochondrial DNA using flanking primers.

31. The forensic method of claim 20, wherein said two or more segments of mitochondrial DNA comprise the entire mitochondrial DNA of said subject.

32. The forensic method of claim 20, wherein said forensic conclusion comprises identification of at least one subject from whom said forensic evidence sample is obtained by comparing said molecular masses of said two or more amplification products with said plurality of known molecular masses in said at least one database.

33. The forensic method of claim 32, wherein said forensic conclusion is the identification of a criminal.

34. The forensic method of claim 32, wherein said forensic conclusion is the identification of a crime victim.

36. The forensic method of claim 20, wherein said forensic conclusion further comprises determining the movement of at least one subject from whom said forensic evidence sample is obtained by mitochondrial DNA analysis of a plurality of forensic evidence samples obtained from a plurality of locations.

37. The forensic method of claim 20, wherein said mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry.

38. The forensic method of claim 20, wherein said at least one database is a Federal Bureau of Investigation mitochondrial DNA database.

47. The forensic method of claim 20, wherein said forensic conclusion comprises identification of a missing person, detection and identification of a known bioagent, detection and identification of an unknown bioagent, elimination of an individual as a crime suspect, identification of an individual as a crime suspect, identification of a location as a crime scene, identification of a location as an accident scene, identification of evidence useful in a court of law, identification of evidence useful in a criminal investigation, or identification of one or more biological samples from a crime scene.

35. The method of 20, further comprising determining the relative amounts of said two or more amplification products from the abundance of mass spectral peaks corresponding to said two or more amplification products.

39. A method of characterizing heteroplasmy of two or more segments of mitochondrial DNA of a subject comprising the steps of:

providing a sample from said subject;

amplifying said two or more segments of mitochondrial DNA from said sample with two or more primer pairs to obtain a plurality of amplification products;

determining molecular masses of said plurality of amplification products by mass spectrometry, without sequencing said plurality of amplification products; and

determining base compositions of said plurality of amplification products thereby characterizing said heteroplasmy.

40. The method of claim 39, wherein said heteroplasmy is selected from the group consisting of length heteroplasmy, nucleotide polymorphism heteroplasmy, or both length heteroplasmy and nucleotide polymorphism heteroplasmy.

41. The method of claim 39, further comprising obtaining a plurality of samples of mitochondrial DNA from said subject at different ages of the individual, wherein the characterization of heteroplasmy indicates the rate of naturally occurring mutations in mitochondrial DNA.

42. The method of claim 39, further comprising comparing said heteroplasmy in said two or more segments of mitochondrial DNA from said sample with at least one database comprising a plurality of base compositions from said two or more segments of mitochondrial DNA from a plurality of subjects with one or more mitochondrial diseases, wherein said comparing correlates said heteroplasmy with the onset of said one or more mitochondrial diseases in a subject.

43. The method of claim 42, wherein said one or more mitochondrial diseases are selected from the group consisting of Alpers Disease, Barth syndrome, Beta-oxidation Defects, Carnitine-Acyl-Carnitine Deficiency, Carnitine Deficiency, Co-Enzyme Q10 Deficiency, Complex I Deficiency, Complex II Deficiency, Complex III Deficiency, Complex IV Deficiency, Complex V Deficiency, COX Deficiency, CPEO, CPT I Deficiency, CPT II Deficiency, Glutaric Aciduria Type II, KSS, Lactic Acidosis, LCAD, LCHAD, Leigh Disease or Syndrome, LHON, Lethal Infantile Cardiomyopathy, Luft Disease, MAD, MCA, MELAS, MERRF, Mitochondrial Cytopathy, Mitochondrial DNA Depletion, Mitochondrial Encephalopathy, Mitochondrial Myopathy, MNGIE, NARP, Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency, Respiratory Chain, SCAD, SCHAD, or VLCAD.

44. The method of claim 39, wherein said mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry.

45. The method of claim 39, wherein said sample from said subject is a forensic evidence sample.