Methods for identifying bioagents
View Patent ↗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. A method of identifying a eukaryotic organism bioagent in a sample, comprising:
(a) amplifying a nucleic acid from said eukaryotic organism bioagent with at least one pair of oligonucleotide primers to form at least one amplification product, wherein one member of said pair of primers hybridizes to a first conserved region of a nucleic acid and the other member of said pair of primers hybridizes to a second conserved region, wherein said conserved regions are present in three or more bioagents, and wherein said first and second conserved regions flank a variable nucleic acid region which varies among at least three bioagents;
(b) analyzing said amplification product or fragments thereof by mass spectrometry to obtain one or more molecular masses;
(c) determining a base composition of said amplification product from said one or more molecular masses of said amplification product or fragments thereof wherein said base compositions identify the number of, but not the nucleic acid gene sequence order of, A residues, C residues, T residues, G residues, and base analogs thereof; and
(d) comparing said one or more base compositions to calculated or measured base compositions of amplification products produced using said pair of oligonucleotide primers, or fragments of said amplification products, thereby identifying said bioagent.
2. The method of claim 1 , wherein said amplification product yields different fragmentation patterns for at least two of said three or more bioagents.
3. The method of claim 1 , wherein said mass spectrometry comprises electrospray ionization-mass spectrometry.
4. The method of claim 1 , wherein said mass spectrometry comprises matrix-assisted laser desorption ionization-mass spectrometry.
5. The method of claim 1 , wherein said mass spectrometry comprises fast atom bombardment ionization-mass spectrometry.
6. The method of claim 1 , wherein said nucleic acid from said bioagent comprises DNA.
7. The method of claim 1 , wherein said nucleic acid from said bioagent comprises RNA.
8. The method of claim 1 , comprising restriction mapping said amplification product or fragments thereof.
9. The method of claim 1 , wherein a molecular mass of said amplification product is about 10 kDa.
10. The method of claim 1 , wherein a molecular mass of said fragment is about 100 Da.
11. The method of claim 1 , wherein said fragments are in a size range of about 50 to 70 base pairs.
12. The method of claim 1 , comprising digesting said amplification product with one or more restriction endonucleases prior to (b).
13. The method of claim 12 , wherein said restriction endonucleases are selected from the group consisting of: BstNI, BsmFI, BfaI, NcoI, RsaI, HpaII, HpyCH4IV, PacI, and Eae1.
14. The method of claim 1 , wherein said eukaryotic organism bioagent is an animal.
15. The method of claim 1 , wherein said animal is a human.
16. The method of claim 1 , wherein said sample is a crime scene sample.
17. The method of claim 1 , wherein said sample is an accident scene sample.