Computational protocol: Oxidative Stress Is Not a Major Contributor to Somatic Mitochondrial DNA Mutations

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Protocol publication

[…] TaqI test primers were designed to flank TaqI sites and amplify products sized between 95 and 250 bp using NCBI's Primer-BLAST program (Primer3, ) (Primer-Blast, National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD). The Primer-BLAST algorithm was also used to optimize primer specificity by testing for mis-priming to related nuclear and mitochondrial sequences. There are four known Drosophila mitochondrial pseudogenes that reside in the nuclear DNA , and these sequences were excluded from primer design to ensure that our primers were specific for mitochondrial sequences. Control primers were designed in the same manner as test primers but do not flank a TaqI site. Because the Drosophila mtDNA has a high A∶T content, control primers were sought at mtDNA sequences with a relatively high G∶C content.We validated the efficiency of control and test primer pairs by measuring the cycle threshold (Ct; the cycle number at which the signal rises above background) using a four-fold dilution series of undigested mtDNA, and plotting Ct vs. dilution (). Theory predicts that product abundance doubles at each cycle of PCR amplification, so the slope of Ct vs. dilution should be 2. Primer pairs that deviated by 15% or more from a slope value of 2 were discarded. Only test and control primers that yielded slope values within 5% of one another were used in our analyses (). Finally, we sequenced the PCR product produced by each primer pair that passed our quality control thresholds to ensure that only the correct target was amplified. [...] Clustal Omega (Cambridge, UK) was used to align human Ogg1 and Drosophila Ogg1 and RpS3 sequences , , and MitoPROT (Munich, Germany) was used to identify N-terminal mitochondrial targeting sequences in Drosophila Ogg1 . Snap Gene software was used to create the Drosophila mtDNA map with TaqI sites (GSL Biotech LLC, Chicago, IL) […]

Pipeline specifications

Software tools Clustal Omega, MITOPROT
Application Protein sequence analysis
Organisms Drosophila melanogaster
Diseases Neoplasms
Chemicals Deoxyguanosine, Superoxides