By Matthew McKenzie (eds.)
This 3rd version is constituted of well-established protocols which are thought of the choicest within the box in addition to new methodologies for mitochondrial DNA research. of Mitochondrial DNA: tools and Protocols describes protocols for detecting mutations in mitochondrial DNA, thoughts to evaluate mitochondrial DNA harm, visualization of mitochondrial DNA in situ, detection of mitochondrial DNA nucleoids in the mitochondria, equipment for examining mitochondrial DNA replication, mitochondrial DNA-encoded protein translation and mitochondrial DNA reproduction quantity, the newest applied sciences for enhancing the mitochondrial genome and strategies for the purification of proteins taken with the replication and transcription of mitochondrial DNA. Written for the Methods in Molecular Biology sequence, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols and tips about troubleshooting and fending off identified pitfalls.
Authoritative and functional, Mitochondrial DNA: equipment and Protocols, 3rd Edition can be beneficial not just for mitochondrial researchers but additionally for scientists learning human ailments the place mitochondrial DNA version has been famous as a tremendous pathogenic issue, together with melanoma and neurodegeneration.
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Extra resources for Mitochondrial DNA: Methods and Protocols
Nat Genet 2:324–329 Meissner C, Bruse P, Mohamed SA, Schulz A, Warnk H, Storm T, Oehmichen M (2008) The 4977 bp deletion of mitochondrial DNA in human skeletal muscle, heart and different areas of the brain: a useful biomarker or more? Exp Gerontol 43:645–652 Kraytsberg Y, Nekhaeva E, Chang C, Ebralidse K, Khrapko K (2004) Analysis of somatic mutations via long-distance single molecule PCR. In: Demidov VV, Broude NE (eds) DNA amplification: current technologies and applications. Horizon, Norfolk, UK, pp 97–110 Kraytsberg Y, Bodyak N, Myerow S, Nicholas A, Ebralidze K, Khrapko K (2009) Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR.
The procedure becomes prohibitively expensive. Increasing the number of wt molecules per well can help, but on the expense of potential loss of some deletions because their bands in the gel become weaker and more difficult to discern from the strong wt bands. Another problem is that identification of deletions involves laborious extraction of deletion bands from gels. Quantitation of Mitochondrial DNA Deletions Via Restriction… 37 To reduce the number of smPCR wells needed to detect a given number of deleted molecules, one can specifically abolish wt molecules by cutting them with a restriction enzyme at a site located between the primers to prevent PCR amplification (Fig.
Trends Genet 20:393–398 9. Guo X, Popadin K, Markuzon N, Orlov YL, Kraytsberg Y, Krishnan KJ, Zsurka G, Turnbull DM, Kunz WS, Khrapko K (2010) Repeats, longevity and the sources of mtDNA deletions: evidence from “deletional spectra”. Trends Genet 26:340–343 10. Damas J, Carneiro J, Gonçalves J, Stewart JB, Samuels DC, Amorim A, Pereira F (2012) Mitochondrial DNA deletions are associated with non-B DNA conformations. Nucleic Acids Res 40:7606–7621 11. Kraytsberg Y, Kudryavtseva E, McKee AC, Geula C, Kowall NW, Khrapko K (2006) Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons.