Patent ID: 9217740
Filing Date: 2015-12-22
Classification: C12Q,G01N

Abstract:
1. A method for identifying a mitochondrial heteroplasmy, comprising: (a) providing at least one eukaryotic cell, wherein the cytoplasm of the at least one eukaryotic cell contains mitochondria, wherein the mitochondria contains mitochondrial DNA, and wherein the mitochondrial DNA is heteroplasmic; (b) lysing the at least one eukaryotic cell with a laser to release the cytoplasm and mitochondria therefrom; (c) isolating and capturing a single mitochondrion from the mitochondria in the cellular cytoplasm released from the at least one eukaryotic cell with optical tweezers; (d) acquiring the single mitochondrion with a femtopipette tip having a pump attached thereto; (e) sonicating and then heating the single mitochondrion with a sonicator and a heat source to release the mitochondrial DNA from the single mitochondrion; (f) providing a series of primers, wherein the primers are specific to pre-selected target regions of the mitochondrial DNA; (g) amplifying the pre-selected target regions of the mitochondrial DNA by polymerase chain reaction; (h) determining the nucleotide sequence of each amplified target region with a sequencing system; (i) comparing the nucleotide sequence of each amplified target region to sequences of mitochondrial DNA that represent known mitochondrial heteroplasmies associated with specific disease states or health conditions of interest; and (j) identifying the presence of a specific mitochondrial heteroplasmy within the single isolated mitochondria based on the comparison of the amplified target region to the sequences of mitochondrial DNA that represent known mitochondrial heteroplasmies.