Human mitochondrial DNA complete amplification and sequencing: A new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co‐amplification

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

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Simultaneous Detection of Human Mitochondrial DNA and Nuclear-Inserted Mitochondrial-origin Sequences (NumtS) using Forensic mtDNA Amplification Strategies and Pyrosequencing Technology

Journal of Forensic Sciences ◽

10.1111/1556-4029.12441 ◽

2014 ◽

Vol 59 (4) ◽

pp. 1064-1073 ◽

Cited By ~ 14

Author(s):

Brittania J. Bintz ◽

Groves B. Dixon ◽

Mark R. Wilson

Keyword(s):

Mitochondrial Dna ◽

Simultaneous Detection ◽

Human Mitochondrial Dna ◽

Mitochondrial Origin ◽

Pyrosequencing Technology

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AT-rich sequences flanking the 5′-end breakpoint of the 4977-bp deletion of human mitochondrial DNA are located between two bent-inducing DNA sequences that assume distorted structure in organello

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/s0027-5107(98)00054-2 ◽

1998 ◽

Vol 403 (1-2) ◽

pp. 75-84 ◽

Cited By ~ 16

Author(s):

Jeng-Horng Hou ◽

Yau-Huei Wei

Keyword(s):

Mitochondrial Dna ◽

Dna Sequences ◽

Human Mitochondrial Dna ◽

Distorted Structure ◽

In Organello

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Integrative taxonomy of a new and highly-diverse genus of onchidiid slugs from the Coral Triangle (Gastropoda, Pulmonata, Onchidiidae)

ZooKeys ◽

10.3897/zookeys.763.21252 ◽

2018 ◽

Vol 763 ◽

pp. 1-111 ◽

Cited By ~ 6

Author(s):

Tricia C. Goulding ◽

Munawar Khalil ◽

Shau Hwai Tan ◽

Benoît Dayrat

Keyword(s):

Mitochondrial Dna ◽

Dna Sequences ◽

Dna Analysis ◽

Nuclear Dna ◽

Comparative Anatomy ◽

Morphological Characters ◽

Integrative Taxonomy ◽

The Philippines ◽

Coral Triangle ◽

Yellow Orange

A new genus of onchidiid slugs,WallaconchisGoulding & Dayrat,gen. n., is described, including ten species. Five species were previously described but known only from the type material:Wallaconchisater(Lesson, 1830),W.graniferum(Semper, 1880),W.nangkauriense(Plate, 1893),W.buetschlii(Stantschinsky, 1907), andW.gracile(Stantschinsky, 1907), all of which were originally classified inOnchidiumBuchannan, 1800. Many new records are provided for these five species, which greatly expand their known geographic distributions. Five species are new:WallaconchisachleitneriGoulding,sp. n.,W.comendadoriGoulding & Dayrat,sp. n.,W.melanesiensisGoulding & Dayrat,sp. n.,W.sinanuiGoulding & Dayrat,sp. n., andW.uncinusGoulding & Dayrat,sp. n.Nine of the tenWallaconchisspecies are found in the Coral Triangle (eastern Indonesia and the Philippines). Sympatry is high, with up to six species found on the island of Bohol (Philippines) and eight species overlapping in northern Sulawesi (Indonesia).Wallaconchisis distinguished from other onchidiids by its bright dorsal colors (red, yellow, orange) but those are extremely variable and not useful for specific identification. Internally, the reproductive system can be used to identify allWallaconchisspecies. The copulatory organs ofWallaconchisspecies are especially diverse compared to other onchidiid genera, and the possible role of reproductive incompatibility in species diversification is discussed. All specimens examined were freshly collected for the purpose of a worldwide revision of the Onchidiidae Rafinesque, 1815. The species are well delineated using DNA sequences and comparative anatomy. Mitochondrial DNA analysis yields thirteen molecular units separated by a large barcode gap, while nuclear DNA yields nine units. By integrating nuclear DNA and mitochondrial DNA with morphology, ten species are recognized. The natural history of each species (e.g., the microhabitat where they are found) is also documented. Nomenclature is addressed thoroughly (the types of all onchidiid species were examined, lectotypes were designated when needed,nomina dubiaare discussed). Morphological characters, transitions to new microhabitats, and diversification processes are discussed in the context of a robust molecular phylogeny.

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