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Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context
Authors:Kurelac Ivana  Lang Martin  Zuntini Roberta  Calabrese Claudia  Simone Domenico  Vicario Saverio  Santamaria Monica  Attimonelli Marcella  Romeo Giovanni  Gasparre Giuseppe
Affiliation:Ostetriche e Pediatriche, Genetica Medica, Pol. Universitario S.Orsola-Malpighi, Bologna, Italy.
Abstract:Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).
Keywords:mtDNA, mitochondrial DNA   MIDD, Maternally Inherited Diabetes and Deafness   NARP, Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa   RFLP, Restriction Fragment Length Polymorphism   F-PCR, Fluorescent PCR   TTGE, Temporal Temperature Gradient Gel Electrophoresis   qRT-PCR, quantitative Real-Time PCR   dHPLC, denaturing High Performance Liquid Chromatography   HRM, High Resolution Melting   indels, small insertions and deletions   MT-ND1, mitochondrially encoded NADH dehydrogenase 1   NumtS, Nuclear Mitochondrial Sequences   QS, Quality Score   PGD, Preimplantation Genetic Diagnosis
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