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

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.

1 - 28 . (canceled) 
     
     
         29 . A system, comprising:
 at least one mass spectrometer configured to determine molecular masses of two or more amplification products, and/or fragments thereof, of two or more segments of mitochondrial DNA from a forensic evidence sample;   at least one database comprising a plurality of known molecular masses from said two or more segments of mitochondrial DNA, and/or fragments thereof, from a plurality of subjects; and,   at least one computer configured to compare said molecular masses of said two or more amplification products, and/or fragments thereof, with said plurality of known molecular masses to reach a forensic conclusion.   
     
     
         30 . The system of  claim 29 , wherein said mass spectrometer is an electrospray ionization mass spectrometer. 
     
     
         31 . The system of  claim 29 , wherein said database is a Federal Bureau of Investigation mitochondrial DNA database. 
     
     
         32 . The system of  claim 29 , wherein said computer is configured to determine relative amounts of said two or more amplification products, and/or fragments thereof, from abundance of mass spectral peaks corresponding to said two or more amplification products, and/or fragments thereof. 
     
     
         33 . The system of  claim 29 , comprising at least one nucleic acid isolation robot configured to isolate nucleic acids from forensic evidence samples. 
     
     
         34 . The system of  claim 29 , wherein said subjects are non-human eukaryotic organisms, fungi, parasites, or protozoa. 
     
     
         35 . The system of  claim 29 , wherein said two or more segments of mitochondrial DNA comprise a portion of a hypervariable region (HVR) of said mitochondrial DNA. 
     
     
         36 . The system of  claim 29 , wherein said hypervariable region comprises at least one of HVR1 or HVR2. 
     
     
         37 . The system of  claim 29 , 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. 
     
     
         38 . The system of  claim 29 , wherein said forensic conclusion comprises an identity of a subject, wherein said computer matches said molecular masses of said amplification products with two or more of said known molecular masses to reach said forensic conclusion. 
     
     
         39 . The system of  claim 38 , wherein said forensic conclusion is the identification of a criminal. 
     
     
         40 . The system of  claim 38 , wherein said forensic conclusion is the identification of a crime victim. 
     
     
         41 . The system of  claim 29 , comprising at least one autosampler operably connected to said computer, which computer is configured to control said autosampler. 
     
     
         42 . The system of  claim 41 , comprising at least one microtiter plate comprising said two or more amplification products, wherein said autosampler is configured to transfer said two or more amplification products to said mass spectrometer. 
     
     
         43 . The system of  claim 29 , wherein said subjects are animals. 
     
     
         44 . The system of  claim 43 , wherein said animals are humans. 
     
     
         45 . A system, comprising:
 at least one mass spectrometer configured to determine molecular masses of two or more amplification products, and/or fragments thereof, of two or more segments of mitochondrial DNA from a forensic evidence sample;   at least one database comprising a plurality of known base compositions from said two or more segments of mitochondrial DNA, and/or fragments thereof, from a plurality of subjects; and,   at least one computer configured to calculate base compositions of said two or more amplification products, and/or fragments thereof, from said molecular masses and to compare said base compositions of said two or more amplification products, and/or fragments thereof, with said plurality of known base compositions to reach a forensic conclusion.   
     
     
         46 . The system of  claim 45 , wherein said mass spectrometer is an electrospray ionization mass spectrometer. 
     
     
         47 . The system of  claim 45 , wherein said database is a Federal Bureau of Investigation mitochondrial DNA database. 
     
     
         48 . The system of  claim 45 , wherein said computer is configured to determine relative amounts of said two or more amplification products, and/or fragments thereof, from abundance of mass spectral peaks corresponding to said two or more amplification products, and/or fragments thereof. 
     
     
         49 . The system of  claim 45 , comprising at least one nucleic acid isolation robot configured to isolate nucleic acids from forensic evidence samples. 
     
     
         50 . The system of  claim 45 , wherein said subjects are non-human eukaryotic organisms, fungi, parasites, or protozoa. 
     
     
         51 . The system of  claim 45 , wherein said two or more segments of mitochondrial DNA comprise a portion of a hypervariable region (HVR) of said mitochondrial DNA. 
     
     
         52 . The system of  claim 45 , wherein said hypervariable region comprises at least one of HVR1 or HVR2. 
     
     
         53 . The system of  claim 45 , 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. 
     
     
         54 . The system of  claim 45 , wherein said forensic conclusion comprises an identity of a subject, wherein said computer matches said base compositions of said amplification products with two or more of said known base compositions to reach said forensic conclusion. 
     
     
         55 . The system of  claim 54 , wherein said forensic conclusion is the identification of a crime victim. 
     
     
         56 . The system of  claim 54 , wherein said forensic conclusion is the identification of a criminal. 
     
     
         57 . The system of  claim 45 , comprising at least one autosampler operably connected to said computer, which computer is configured to control said autosampler. 
     
     
         58 . The system of  claim 57 , comprising at least one microtiter plate comprising said two or more amplification products, wherein said autosampler is configured to transfer said two or more amplification products to said mass spectrometer. 
     
     
         59 . The system of  claim 45 , wherein said subjects are animals. 
     
     
         60 . The system of  claim 59 , wherein said animals are humans. 
     
     
         61 . A system, comprising:
 at least one mass spectrometer configured to determine molecular masses of two or more amplification products of two or more segments of mitochondrial DNA from a subject; and,   at least one computer configured to determine base compositions of said two or more amplification products from said molecular masses and to characterize heteroplasmy of said two or more segments of mitochondrial DNA from said base compositions.   
     
     
         62 . The system of  claim 61 , wherein said mass spectrometer is an electrospray ionization mass spectrometer. 
     
     
         63 . The system of  claim 61 , wherein said computer is configured to characterize length heteroplasmy, nucleotide polymorphism heteroplasmy, or both length heteroplasmy and nucleotide polymorphism heteroplasmy. 
     
     
         64 . The system of  claim 61 , wherein said computer is configured to characterize a rate of naturally occurring mutations in mitochondrial DNA. 
     
     
         65 . The system of  claim 61 , comprising at least one nucleic acid isolation robot configured to isolate nucleic acids from forensic evidence samples. 
     
     
         66 . The system of  claim 61 , comprising 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. 
     
     
         67 . The system of  claim 66 , wherein said computer is configured to compare said base compositions of said two or more amplification products with one or more base compositions of said database to correlate said heteroplasmy with an onset of said one or more mitochondrial diseases in said subject. 
     
     
         68 . The system of  claim 66 , 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, and VLCAD. 
     
     
         69 . The system of  claim 61 , comprising at least one autosampler operably connected to said computer, which computer is configured to control said autosampler. 
     
     
         70 . The system of  claim 69 , comprising at least one microtiter plate comprising said two or more amplification products, wherein said autosampler is configured to transfer said two or more amplification products to said mass spectrometer.