Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Elimination of rNMPs from mitochondrial DNA has no effect on its stability
Figure Lengend Snippet: Deletion of SAMHD1 does not affect mtDNA stability. ( A ) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-wk-old (adult), 1-y-old (old adult), and 2-y-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The P values were calculated using Welch’s t test; ns, nonsignificant. ( B ) DNA isolated from embryos and from the TA muscle of pups, adults, 1-y-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. One undigested sample is shown for comparison (Uncut). ( C ) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. ( D ) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. ( E ) The median length of the untreated mtDNA in samples from D is indicated by a horizontal line. The two groups were compared using Welch’s t test (ns, nonsignificant; n = 4). ( F ) The length difference between untreated and alkali-treated mtDNAs shown in D was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The P value of the statistically significant difference between the two groups was calculated by Welch’s t test; n = 4. ( G ) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. ( H ) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also SI Appendix , Fig. S5 .
Article Snippet: MtDNA Deletion Analysis by Long-Range PCR.The Expand Long Template PCR system (Roche) with forward and reverse primers at nt 2,478 to 2,512 and nt 1,933 to 1,906, respectively, was used to amplify an ∼15,800-base pair fragment of mouse mtDNA from 25 ng of total DNA.
Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Isolation, Polymerase Chain Reaction