ABSTRACT
Mitochondria, the power house of cells, are important organelles in eukaryotic cells. Having their own unique and complete DNA (mtDNA) and genetic system, mitochondria play an essential role in cellular energy metabolism, intracel?lular signaling and apoptotic pathways, as well as many other biological functions, which are closely related with cellular met?abolic network. A disruption of mitochondrial genes can therefore result in mitochondrial dysfunction and human diseases, thus they have been widely used in molecular biology, development biology, genetics, forensic identification and clinical diag?nosis. Consequently, sequencing mitochondrial genome has shown great significance in mitochondrial structure and function research. In this review, research progress in mitochondrial genome sequencing method is summarized, mainly focusing on Sanger sequencing, long-PCR and next-generation sequencing. Also rolling circle amplification and indirect sequencing of mtDNA are reviewed. The ambiguities caused by numts in indirect sequencing are mentioned and resolved.
ABSTRACT
Molecular tools have been used in taxonomy for the purpose of identification and classification of living organisms. Among these, a short sequence of the mitochondrial DNA, popularly known as DNA barcoding, has become very popular. However, the usefulness and dependability of DNA barcodes have been recently questioned because mitochondrial pseudogenes, non-functional copies of the mitochondrial DNA incorporated into the nuclear genome, have been found in various taxa. When these paralogous sequences are amplified together with the mitochondrial DNA, they may go unnoticed and end up being analyzed as if they were orthologous sequences. In this contribution the different points of view regarding the implications of mitochondrial pseudogenes for entomology are reviewed and discussed. A discussion of the problem from a historical and conceptual perspective is presented as well as a discussion of strategies to keep these nuclear mtDNA copies out of sequence analyzes.
Ferramentas moleculares têm sido utilizadas para os estudos referentes à identificação e classificação dos organismos vivos. Entre estes, uma curta sequência do DNA mitocondrial, popularmente conhecida como DNA barcoding, tornou-se muito popular. No entanto, a utilidade e confiabilidade dos códigos de barras de DNA têm sido recentemente questionadas porque pseudogenes mitocondriais, cópias não-funcionais do DNA mitocondrial incorporados ao genoma nuclear, foram encontrados em vários táxons. Quando estas sequências parálogas são amplificadas juntamente com o DNA mitocondrial, podem passar despercebidas e acabam sendo analisadas como se fossem seqüências ortólogas. Nesta contribuição objetivou-se revisar e discutir os diferentes pontos de vista sobre as implicações de pseudogenes mitocondriais para entomologia. Discutimos também o problema através de uma perspectiva histórica e conceitual, abordando estratégias para eliminar ou evitar a presença dessas cópias nucleares em meio às sequências funcionais de DNA.