Quantifying Mitochondrial Genome Integrity

qPCR was carried out on an Applied Biosystems 7300 Real Time PCR machine using iTaq SYBR Green Supermix with ROX (Bio-Rad). 1 uL of genomic DNA (diluted 1:5 in water) was used for each qPCR analysis. To estimate the total amount of mtDNA in a sample (both intact and deletion-bearing), control primers were designed to amplify a 102 bp region of the small ribosomal RNA subunit that displayed no evidence of heteroplasmy. A second set of primers was designed to amplify 101 bp in the 5' end of the ND5 gene, within the deleted region. This product is not expected to amplify only in intact genomes. qPCR data were analyzed to estimate the abundances of the two genome types using the linear regression approach offered by the LinRegPCR software [40 (link)]. ND5 locus products were found to amplify more efficiently than ribosomal RNA products; thus, all ND5 qPCR values were normalized to account for this disparity. Individual deletion genotype proportions were calculated by dividing the estimated abundance of intact genomes by that of the total mitochondrial genomes, and then subtracting that number from one. Four individual L1-stage nematodes were analyzed per natural isolate – the same nematode genomic DNA samples used for conventional PCR assays.

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Howe D.K, & Denver D.R. (2008). Muller's Ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution. BMC Evolutionary Biology, 8, 62.

Publication 2008

Assays Deletion Gene Genomic Genotype Heteroplasmy Mitochondrial genomes Mtdna Nematode Primers Real time pcr Ribosomal rna Small ribosomal subunit Sybr green

Corresponding Organization :

Other organizations : Oregon State University, Biocom

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Abundances of intact genomes and total mitochondrial genomes
Individual deletion genotype proportions

control variables

Genomic DNA samples from 4 individual L1-stage nematodes per natural isolate
QPCR analysis using an Applied Biosystems 7300 Real Time PCR machine
Use of iTaq SYBR Green Supermix with ROX (Bio-Rad)
Use of 1 uL of genomic DNA (diluted 1:5 in water) for each qPCR analysis
Design of control primers to amplify a 102 bp region of the small ribosomal RNA subunit that displayed no evidence of heteroplasmy
Design of a second set of primers to amplify 101 bp in the 5' end of the ND5 gene, within the deleted region
Normalization of ND5 locus qPCR values to account for their more efficient amplification compared to ribosomal RNA products

controls

Positive control: None mentioned
Negative control: None mentioned

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