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Validating the Optical Tweezers Method for Trapping a Single Mitochondrion by Utilizing Quantitative Real‐Time PCR
Author(s) -
Knipe Ashley L,
Boire Nicholas,
Tobin Lisa,
Reiner Joseph,
Kishore Rani,
Helmerson Kristian,
Levin Barbara,
Deckman Koren Holland
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a661-d
Subject(s) - heteroplasmy , mitochondrial dna , taqman , amplicon , mitochondrion , optical tweezers , biology , microbiology and biotechnology , genome , plasmid , real time polymerase chain reaction , genetics , gene , polymerase chain reaction , computational biology , physics , optics
In previous studies, a C/T heteroplasmy, located at nucleotide 12,071 and unique to the mitochondrial genome of HL‐60 cells, was detected in a single mitochondrion. Former studies used optical tweezers (5 watt fiber IPG Photo Optical laser and the Axiovert 100 M fluorescent microscope) and standard amplification methods using NIST SRM 2392‐I primer set 43 to trap and sequence samples of single mitochondria. Although the experimental conditions tried to eliminate potential contaminants, further methods must be used to validate these results. Thus, it must be determined whether a single mitochondrion was indeed trapped and amplified. To address this question, a quantitative real‐time PCR method was developed. Primers and TaqMan® probe within tRNA‐asn gene and an external plasmid control (pDrive; Qiagen, Inc.) with a larger amplicon flanking the tRNA‐asn gene were designed. Samples containing one or two trapped mitochondria were then amplified using the ABI Prism 7000 and TaqMan® Fast Universal PCR Master Mix (ABI). Our initial studies suggest that the samples containing one or two mitochondria show a mtDNA quantity ratio of almost 1:2. With replicates, one can establish a trend of mtDNA quantity in these samples. With the external plasmid control, one can establish mtDNA genome copy numbers per mitochondria and per cell. Our steps toward these goals will be discussed.