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

Abstract

To date, there are no published primers to amplify the entire mitochondrial DNA (mtDNA) that completely prevent the amplification of nuclear DNA (nDNA) sequences of mitochondrial origin. The main goal of this work was to design, validate and describe a set of primers, to specifically amplify and sequence the complete human mtDNA, allowing the correct interpretation of mtDNA heteroplasmy in healthy and pathological samples. Validation was performed using two different approaches: (i) Basic Local Alignment Search Tool and (ii) amplification using isolated nDNA obtained from sperm cells by differential lyses. During the validation process, two mtDNA regions, with high similarity with nDNA, represent the major problematic areas for primer design. One of these could represent a non-published nuclear DNA sequence of mitochondrial origin. For two of the initially designed fragments, the amplification results reveal PCR artifacts that can be attributed to the poor quality of the DNA. After the validation, nine overlapping primer pairs to perform mtDNA amplification and 22 additional internal primers for mtDNA sequencing were obtained. These primers could be a useful tool in future projects that deal with mtDNA complete sequencing and heteroplasmy detection, since they represent a set of primers that have been tested for the non-amplification of nDNA.

Mitochondrial DNA patterns in the Iberian Northern plateau: Population dynamics and substructure of the Zamora province

“…For each sample, the mtDNA hypervariable region I (HVRI) was amplified and sequenced, between positions 16,024 and 16,399, and coding region informative polymorphisms (mtDNA positions: 1,018;3,010;4,580;6,776;7,028;8,994;9,055;10,238;11,251;12,308;12,705;14,766) for haplogroup (Hg) assignment were analyzed by PCR-RFLP as previously detailed by Santos et al (2003Santos et al ( , 2004. Moreover, positions 10,400 and 10,873 were also analyzed; the fragment enclosing these mtDNA positions was amplified and sequenced using primers and conditions described by Ramos et al (2009). Sequence reactions were carried out using the sequencing kit BigDye Terminator v.3 (Applied Biosystems) according to the manufacturer's specifications and run in an ABI 3130XL sequencer (Genomics Unit, Universitat Autònoma de Barcelona).…”

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants

“…Nine overlapping primer pairs were used for mtDNA amplification with 62 corresponding overlapping internal sequencing primers for mtDNA sequencing as previously described. 19,20 Bidirectional cycle sequencing was performed using BigDye Terminator v3.1 cycle sequencing kit (Life Technologies), and amplified, targeted genetic regions electrophoresed on an ABI PRISM 3730 Genetic Analyzer (Life Technologies).…”

Echoes from Sepharad: signatures on the maternal gene pool of crypto-Jewish descendants

“…33 Sampling criteria and collection method, as well as DNA extraction, are described in Nogueiro et al 27 The study was approved by the Ethics Committee of the University of Porto (N102/CEUP/2012) and appropriate informed consent was required from all subjects. Full mtDNA sequences were obtained using the protocol described in Ramos et al 34,35 Sequences were aligned against the revised Cambridge Reference Sequence GenBank accession number NC_012920.1 36 using Genious software version 5.5.8 (http://www.geneious.com), variants were annotated following the HGVS rules (http://www.hgvs.org/mutnomen/). Haplogroups were classified following the updated mtDNA phylogeny, PhyloTree, mtDNA tree Build 16 37 (http://www.phylotree.org/) and assigned haplotypes were submitted to the EMPOP database 38 (http://empop.org/).…”