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

• US 9,752,184 B2
• Filed: 08/11/2014
• Issued: 09/05/2017
• Est. Priority Date: 03/02/2001
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Griffey, Richard H., Sampath, Rangarajan, Hofstadler, Steven A., McNeil, John, Crooke, Stanley T., Blyn, Lawrence B., Hall, Thomas A., Jiang, Yun, Hannis, James C., White, Neill K., Samant, Vivek, Eshoo, Mark W., Drader, Jared J.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US14/456,806
• Publication Number:
  US 20150031117A1
• Time in Patent Office: 1,121 Days
• Field of Search: None
• 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 system, comprising:

a) a nucleic acid purification component for purifying a nucleic acid from an unknown bioagent;

b) a nucleic acid amplification component for amplifying a target nucleic acid sequence region from an unknown bioagent to generate an amplicon comprising said target nucleic acid sequence region, and at least one pair of purified oligonucleotide primers that hybridizes to said at least one target sequence region of a plurality of known bioagents, said target nucleic acid sequence region comprising two conserved regions that are hybridizable with said primer pair and that flank a variable region that uniquely varies between a plurality of known bioagents;

c) a base composition determination component for determining a base composition of said target nucleic acid sequence region comprising a computer processor configured to determine the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof of said amplicon;

d) a base composition identification component comprising a database of base compositions from a plurality of known bioagents wherein said base compositions identify the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof, said database comprising a plurality of measured base compositions of amplification products of known bioagents calculated or obtained by amplification of at least one target nucleic acid sequence region of a known bioagent nucleic acid gene sequence using a primer pair that hybridizes to said at least one target nucleic acid sequence region of a plurality of known bioagents said target nucleic acid sequence region comprising two conserved regions that are hybridizable with said primer pair and that flank a variable region that uniquely varies between a plurality of known bioagents, wherein said computer processor compares said base compositions from said plurality of known bioagents to a base composition of an unknown bioagent to determine the identity of said unknown bioagent; and

e) a computer program on a computer readable medium configured to direct said computer processor to coordinate the operation of said nucleic acid purification component, said nucleic acid amplification component, said base composition determination component, and said base composition identification component.

2. The system of claim 1, wherein said nucleic acid purification component comprises one or more buffer manipulations, one or more salt manipulations, one or more thermal manipulations, one or more pH manipulations, one or more mechanical manipulations, one or more centrifugation manipulations, or one or more magnetic manipulations.

3. The system of claim 1, wherein said nucleic acid amplification component comprises a thermocycler.

4. The system of claim 1, wherein the amplification step is quantitative.

5. The system of claim 1, wherein said nucleic acid amplification component comprises one or more salts, one or more buffers, one or more dNTPs, or one or more enzymes.

6. The system of claim 1, wherein said database of base compositions comprises at least 10 base compositions.

7. The system of claim 1, wherein said database of base compositions comprises at least 50 base compositions.

8. The system of claim 1, wherein said database of base compositions comprises at least 100 base compositions.

9. The system of claim 1, wherein said database of base compositions comprises at least 500 base compositions.

10. The system of claim 1, wherein said database of base compositions comprises at least 1000 base compositions.

11. The system of claim 1, wherein said plurality of known bioagents comprises bioagents that differ by genus, species, sub-species, strain, sub-type or nucleotide polymorphism.

12. The system of claim 1, wherein said plurality of known bioagents comprises one or more viral bioagents, one or more bacterial bioagents, one or more fungal bioagents, one or more protozoal bioagents, one or more parasitic bioagents, one or more mammalian bioagents, or one or more human bioagents.

13. The system of claim 1, wherein said database of base compositions comprises the number but not the nucleic acid sequence order of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof.

14. The system of claim 1, wherein said at least one pair of purified oligonucleotide primers hybridizes to at least one target sequence region within a gene.