title: The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation.
abstract: BACKGROUND
  Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results.
  
  
  RESULTS
  Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (rho0) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences.
  
  
  CONCLUSIONS
  These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.
authors.affiliation: Genesis Genomics Inc, 1294 Balmoral Street, Thunder Bay, Ontario, P7B 5Z5, Canada. Ryan.Parr@genesisgenomics.com
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Parr
authors.forename: Jennifer
authors.initials: J
authors.lastname: Maki
authors.forename: Brian
authors.initials: B
authors.lastname: Reguly
authors.forename: Gabriel D
authors.initials: GD
authors.lastname: Dakubo
authors.forename: Andrea
authors.initials: A
authors.lastname: Aguirre
authors.forename: Roy
authors.initials: R
authors.lastname: Wittock
authors.forename: Kerry
authors.initials: K
authors.lastname: Robinson
authors.forename: John P
authors.initials: JP
authors.lastname: Jakupciak
authors.forename: Robert E
authors.initials: RE
authors.lastname: Thayer
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D004272:DNA, Mitochondrial; D005260:Female; D018628:Gene Dosage; D030342:Genetic Diseases, Inborn / Q000175:diagnosis; D015894:Genome, Human; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012516:Osteosarcoma / Q000235:genetics; D010920:Placenta / Q000378:metabolism; D011468:Prostatectomy; D011544:Pseudogenes; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
chemical_list: D004272:DNA, Mitochondrial