Biomaterial Database

Measurement of mitochondrial dNTP pools. 2012

Ramon Martí, and Beatriz Dorado, and Michio Hirano

Laboratori de Patologia Mitocondrial, Institut de Recerca Hospital Universitari Vall D'Hebron, Universitat Autonoma de Barcelona, Barcelona, Spain.

Because deoxyribonucleoside triphosphates (dNTPs) are the critical substrates for DNA replication and repair, dNTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.

UI	MeSH Term	Description	Entries
D008099	Liver	A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.	Livers
D008928	Mitochondria	Semiautonomous, self-reproducing organelles that occur in the cytoplasm of all cells of most, but not all, eukaryotes. Each mitochondrion is surrounded by a double limiting membrane. The inner membrane is highly invaginated, and its projections are called cristae. Mitochondria are the sites of the reactions of oxidative phosphorylation, which result in the formation of ATP. They contain distinctive RIBOSOMES, transfer RNAs (RNA, TRANSFER); AMINO ACYL T RNA SYNTHETASES; and elongation and termination factors. Mitochondria depend upon genes within the nucleus of the cells in which they reside for many essential messenger RNAs (RNA, MESSENGER). Mitochondria are believed to have arisen from aerobic bacteria that established a symbiotic relationship with primitive protoeukaryotes. (King & Stansfield, A Dictionary of Genetics, 4th ed)	Mitochondrial Contraction,Mitochondrion,Contraction, Mitochondrial,Contractions, Mitochondrial,Mitochondrial Contractions
D002478	Cells, Cultured	Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.	Cultured Cells,Cell, Cultured,Cultured Cell
D003854	Deoxyribonucleotides	A purine or pyrimidine base bonded to a DEOXYRIBOSE containing a bond to a phosphate group.	Deoxyribonucleotide
D004259	DNA-Directed DNA Polymerase	DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.	DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D005347	Fibroblasts	Connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules.	Fibroblast
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D012867	Skin	The outer covering of the body that protects it from the environment. It is composed of the DERMIS and the EPIDERMIS.
D051379	Mice	The common name for the genus Mus.	Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus