Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification.
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.
Departament BABVE, Unitat d'Antropologia Biològica, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. amanda.ramosr@campus.uab.cat
Amanda
A
Ramos
Cristina
C
Santos
Luis
L
Alvarez
Ramon
R
Nogués
Maria Pilar
MP
Aluja
D017076:Computer-Aided Design; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D054629:Genome, Mitochondrial; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D004272:DNA, Mitochondrial