title: Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context.
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).
authors.affiliation: Ostetriche e Pediatriche, Genetica Medica, Pol. Universitario S.Orsola-Malpighi, Bologna, Italy.
authors.forename: Ivana
authors.initials: I
authors.lastname: Kurelac
authors.forename: Martin
authors.initials: M
authors.lastname: Lang
authors.forename: Roberta
authors.initials: R
authors.lastname: Zuntini
authors.forename: Claudia
authors.initials: C
authors.lastname: Calabrese
authors.forename: Domenico
authors.initials: D
authors.lastname: Simone
authors.forename: Saverio
authors.initials: S
authors.lastname: Vicario
authors.forename: Monica
authors.initials: M
authors.lastname: Santamaria
authors.forename: Marcella
authors.initials: M
authors.lastname: Attimonelli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Romeo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Gasparre
mesh_terms: D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial