Methods For Rapid Forensic Analysis Of Mitochondrial DNA

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.

What is claimed is: 
     
         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 entries 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 testing each member of said plurality of restriction fragments; and   comparing said experimentally tested members with 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 1  further comprising:
 populating said 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; 
 experimentally determining the base composition of each member of said plurality of restriction fragments from said experimentally determined molecular masses of each member of said plurality of restriction fragments; 
 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. 
 
     
     
         5 . The method of  claim 1  wherein said amplifying step comprises polymerase chain reaction. 
     
     
         6 . The method of  claim 5  wherein said polymerase chain reaction is catalyzed by a polymerase enzyme whose function is modified relative to a native polymerase. 
     
     
         7 . The method of  claim 6  wherein said modified polymerase enzyme is exo(−) Pfu polymerase. 
     
     
         8 . The method of  claim 6  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. 
     
     
         9 . The method of  claim 1  wherein said amplifying step comprises ligase chain reaction or strand displacement amplification. 
     
     
         10 . The method of  claim 1  wherein said database is a human mtDNA population database. 
     
     
         11 . The method of  claim 1  wherein said testing comprises mass determination by mass spectrometry. 
     
     
         12 . The method of  claim 11  wherein said mass spectrometry is ESI-TOF mass spectrometry. 
     
     
         13 . The method of  claim 1  further comprising repeating all steps of the method for at least one additional region of mitochondrial DNA. 
     
     
         14 . The method of  claim 1  wherein said mitochondrial DNA is human mitochondrial DNA. 
     
     
         15 . The method of  claim 1  wherein said mitochondrial DNA is animal mitochondrial DNA. 
     
     
         16 . The method of  claim 1  wherein said mitochondrial DNA is fungal, parasitic, or protozoan DNA. 
     
     
         17 . The method of  claim 1  wherein said amplified DNA is digested directly without purification. 
     
     
         18 . The method of  claim 1  wherein said sample of mitochondrial DNA is obtained from saliva, hair, blood, or nail. 
     
     
         19 . The method of  claim 1  wherein said plurality of restriction fragments are up to about 150 base pairs in length.