Large DNA deletions drive premature aging in mitochondrial mutator mice Marc Vermulst 1 , Jonathan Wanagat 2 , Gregory C. Kujoth 3 , Jason H. Bielas 1 , Peter S. Rabinovitch 1 , Tomas A. Prolla 3 , Lawrence A. Loeb 1 1 Department of Pathology, University of Washington, Seattle, 91895 Washington, USA. 2 Department of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA 91895, USA 3 Departments of Genetics and Medical Genetics, University of Wisconsin, Madison, WI 53706, USA. Supplementary Material Materials and Methods MtDNA deletion detection. Deletions were PCR-amplified and quantified using our recently described RMC-methodology 1 with minor alterations (Fig. S1). Deleted mtDNA molecules were quantified by single molecule amplification according to previously published protocols 2 , or in triplicate using standard comparative qPCR-methodology. Primer sequences are listed in table S1. PCR conditions PCR reactions were performed using a DNA Engine Opticon ® Monitor 2, a continuous fluorescence detection system from BioRad, and amplicons were quantified with Stratagene’s Brilliant ® SYBR ® Green qPCR Mastermix. QPCR reactions were performed in 25 l reactions containing 1xBrilliant ® SYBR ® Green qPCR Mastermix from Stratagene, 20 pM forward and reverse primers, and 2 u uracil DNA glycosylase (UDG). The samples were amplified as follows: UDG incubation at 37 °C for 10 min, 95 °C for 10 min, and then 45 cycles of 95 °C for 30 sec, 60 °C for 1 min and 72 °C for 1.5 min. Samples were held at 72 °C for 5 min and stored at -20 °C. Histochemistry. Cytochrome oxidase staining solutions were prepared in 10ml aliquots containing 750mg sucrose, 5mg DAB, 10mg cytochrome c. 1 ml 10xcatalase, and 9ml 50mM sodium phosphate buffer pH 7.4. Succinate dehydrogenase staining solutions were prepared in 10 ml aliquots containing
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Large DNA deletions drive premature aging in mitochondrial mutator mice Marc Vermulst1, Jonathan Wanagat2, Gregory C. Kujoth3, Jason H. Bielas1, Peter S. Rabinovitch1, Tomas A. Prolla3, Lawrence A. Loeb1 1Department of Pathology, University of Washington, Seattle, 91895 Washington, USA. 2Department of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA 91895, USA 3Departments of Genetics and Medical Genetics, University of Wisconsin, Madison, WI 53706, USA. Supplementary Material Materials and Methods
MtDNA deletion detection. Deletions were PCR-amplified and quantified using
our recently described RMC-methodology1 with minor alterations (Fig. S1).
Deleted mtDNA molecules were quantified by single molecule amplification
according to previously published protocols2, or in triplicate using standard
comparative qPCR-methodology. Primer sequences are listed in table S1.
PCR conditions PCR reactions were performed using a DNA Engine Opticon®
Monitor 2, a continuous fluorescence detection system from BioRad, and
amplicons were quantified with Stratagene’s Brilliant® SYBR® Green qPCR
Mastermix. QPCR reactions were performed in 25 � l reactions containing
1xBrilliant® SYBR® Green qPCR Mastermix from Stratagene, 20 pM forward and
reverse primers, and 2 u uracil DNA glycosylase (UDG). The samples were
amplified as follows: UDG incubation at 37 °C for 10 min, 95 °C for 10 min, and
then 45 cycles of 95 °C for 30 sec, 60 °C for 1 min and 72 °C for 1.5 min.
Samples were held at 72 °C for 5 min and stored at -20 °C.
Histochemistry. Cytochrome oxidase staining solutions were prepared in 10ml
aliquots containing 750mg sucrose, 5mg DAB, 10mg cytochrome c. 1 ml
10xcatalase, and 9ml 50mM sodium phosphate buffer pH 7.4. Succinate
dehydrogenase staining solutions were prepared in 10 ml aliquots containing
100mg sodium succinate, 10mg NBT, 0.2mg phenazine methosulphate, and
10ml Tris-HCl, pH7.4. Frozen tissue sections were incubated with cytochrome
oxidase staining solution at room temperature for either 15 (heart), 35
(duodenum), or 60 minutes (brain), washed with 1xPBS, and incubated with
succinate dehydrogenase staining solution for 3 (heart), 10 (duodenum) or 30
minutes (brain) at 37°C. Samples where then washed with 1xPBS and either
coverslipped or washed in 70% ethanol.
Microdissection. Microdissection was performed on a PALM MicroBeam
System from P.A.L.M. Microlaser Technologies AG and Zeiss. Laser dissected
samples were caught in opaque adhesive caps from P.A.L.M. Microlaser
Technologies, inspected under a microscope, and lysed in a 3 � l reaction
containing 0.4mg/ml ProK, 0.5% SDS and 10mM EDTA at 46°C.
Tissue homogenization and organelle separation. Animals were sacrificed
and all tissues were harvested within 5 minutes of death. All animals were
cared for according to approved guidelines by the University of Washington and
the University of Wisconsin. Tissues were sliced in pieces with a scalpel and
rinsed in 1xPBS before homogenization in a Dounce-type glass homogenizer
with 25 firm strokes of a hand driven glass pestle. Homogenization buffer
isolated mtDNA deletions in WT, PolgA+/mut and PolgAmut/mut mice. Breakpoints
are depicted by vertical red lines. Some deletions were associated with
mutations around the breakpoint. If present, these are listed on the far right.
1. Bielas, J.H. & Loeb, L.A. Quantification of random genomic mutations. Nat
Methods 2, 285-90 (2005). 2. Vermulst, M. et al. Mitochondrial point mutations do not limit the natural lifespan
of mice. Nat Genet 39, 540-3 (2007). 3. Copeland. Mitochondrial DNA, methods and protocols. Methods in Molecular
Biology 197(2002). 4. Greaves, L.C. et al. Mitochondrial DNA mutations are established in human
colonic stem cells, and mutated clones expand by crypt fission. Proc Natl Acad Sci U S A 103, 714-9 (2006).
5. Wanagat, J., Cao, Z., Pathare, P. & Aiken, J.M. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. Faseb J 15, 322-32 (2001).
6. Taylor, R.W. et al. Mitochondrial DNA mutations in human colonic crypt stem cells. J Clin Invest 112, 1351-60 (2003).