Isolation and characterization of a single mitochondrion

Abstract

A system for identifying mitochondrial heteroplasmy within eukaryotic cells is provided. This system includes means for isolating and capturing a single mitochondrion from at least one eukaryotic cell, wherein the means for isolating and capturing a single mitochondrion further includes optical tweezers or a similar optical technology; means for analyzing the isolated and captured mitochondrion, wherein the means for analyzing the isolated and captured mitochondrion further includes a DNA amplification system and a sequencing system for amplifying and sequencing DNA extracted from the mitochondrion; means for identifying at least one mitochondrial heteroplasmy of interest; and means for using the DNA amplification and DNA sequencing systems to determine the presence or absence of the mitochondrial heteroplasmy within the eukaryotic cell from which the mitochondrion was obtained.

1 . A system for characterizing a single mitochondrion, comprising:
 (a) optical means for isolating and capturing a single mitochondrion from a eukaryotic cell; and   (b) amplification and sequencing means for analyzing the isolated and captured mitochondrion.   
     
     
         2 . The system of  claim 1 , further comprising:
 (a) means for identifying at least one target mitochondrial heteroplasmy; and   (b) using the DNA amplification system and the DNA sequencing system to determine the presence or absence of the target mitochondrial heteroplasmy within the eukaryotic cell.   
     
     
         3 . The system of  claim 2 , wherein the mitochondrial heteroplasmy is associated with at least one specific disease state or condition of interest. 
     
     
         4 . The system of  claim 1 , wherein the eukaryotic cell is of the HL-60 human cell line. 
     
     
         5 . The system of  claim 1 , wherein the means for isolating and capturing a single mitochondrion further comprises labeling the mitochondrion with a fluorescent dye. 
     
     
         6 . The system of  claim 1 , wherein the means for isolating and capturing a single mitochondrion further comprises a micropipette. 
     
     
         7 . The system of  claim 1 , wherein the DNA amplification system further comprises polymerase chain reaction. 
     
     
         8 . A system for identifying mitochondrial heteroplasmy within a eukaryotic cell, comprising:
 (a) means for isolating and capturing a single mitochondrion from a eukaryotic cell, wherein the means for isolating and capturing a single mitochondrion further includes means for creating and operating optical tweezers;   (b) means for analyzing the isolated and captured mitochondrion, wherein the means for analyzing the isolated and captured mitochondrion further includes a DNA amplification system and a DNA sequencing system;   (c) means for identifying at least one mitochondrial heteroplasmy; and   (d) means for using the DNA amplification and sequencing system to determine the presence or absence of the mitochondrial heteroplasmy within the eukaryotic cell.   
     
     
         9 . The system of  claim 8 , wherein the mitochondrial heteroplasmy is associated with at least one specific disease state or condition of interest. 
     
     
         10 . The system of  claim 8 , wherein the eukaryotic cell is of the HL-60 human cell line. 
     
     
         11 . The system of  claim 8 , wherein the optical means further include optical tweezers that include a Nd:YAG fiber laser operating at about 1064 nm with about 1 W of power. 
     
     
         12 . The system of  claim 8 , wherein the means for isolating and capturing a single mitochondrion further comprises labeling the mitochondrion with a fluorescent dye. 
     
     
         13 . The system of  claim 8 , wherein the means for isolating and capturing a single mitochondrion further comprises a micropipette. 
     
     
         14 . The system of  claim 8 , wherein the DNA amplification system further comprises polymerase chain reaction. 
     
     
         15 . A method for characterizing a single mitochondrion, comprising:
 (a) providing at least one eukaryotic cell containing mitochondria;   (b) treating the at least one eukaryotic cell to release the cytoplasm therefrom, wherein the cytoplasm contains cellular organelles;   (c) isolating a single mitochondrion from the cellular organelles using optical tweezers;   (d) capturing the single mitochondrion isolated with the optical tweezers;   (e) treating the single mitochondrion to extract mtDNA therefrom;   (f) selecting a target region within the mtDNA;   (g) amplifying the target region of the mtDNA; and   (f) sequencing the target region of the mtDNA to determine the nucleotide sequence thereof.   
     
     
         16 . The method of  claim 15 , further comprising comparing the sequence of the target region against a known mitochondrial heteroplasmy for determining the presence or absence of the mitochondrial heteroplasmy within the eukaryotic cell. 
     
     
         17 . The method of  claim 15 , wherein the optical tweezers further include a Nd:YAG fiber laser. 
     
     
         18 . The method of  claim 15 , further comprising the step of labeling the mitochondria with a fluorescent dye. 
     
     
         19 . The method of  claim 15 , wherein the target region includes a least one mutation in the mtDNA, and wherein the at least one mutation is associated with at least one disease state or condition of interest. 
     
     
         20 . The method of  claim 15 , wherein amplifying the target region of DNA further includes the user of polymerase chain reaction.