D-loop sequences retrieved from Canis lupus familiaris mitochondrial genome

Canine mitochondrial genome is built of 16727 bp. Non-coding control region (mtCR), called also D-loop, begins with 15458 nucleotide and ends with 16727 nucleotide. The length of this fragment is 1270 bp (Kim et al., 1998). D-loop region is responsible for replication and transcription of mitochondrial DNA. Mutations that occur within it may cause irregularity in those processes and affect electron transport chain. This may lead to increase of level of reactive oxygen species (ROS) in cell. Higher level of ROS can cause new mutations and lead to neoplastic changes (Ślaska et al., 2015). The aim of the conducted investigations was to identify differences in the D-loop nucleotide sequence between neoplastic tissue, normal tissue, and blood and to determine their correlation with the type of cancer in dogs. DNA was isolated with the use of an automated nucleic acid extraction system – QIACube (Qiagen, Hilden, Germany). DNA was extracted from tumor tissues and their matched normal tissues with the DNeasy Blood & Tissue Kit (Qiagen). DNA samples were assessed quantitatively and qualitatively by electrophoretic separation in agarose gel and spectrophotometrically by measurements of sample absorbance in a BioPhotometer (Eppendorf, Hamburg, Germany) spectrophotometer. Amplification of the D-loop, was performed using the PCR technique. Primers used in the analysis, comprising an mtDNA fragment between nucleotide 15341–15804 (LF 5'- TTACCTTGGTCTTGTAAACC; HR 5'-CTGAAGTAAGAACCAGATGCC), were found in literature reports (van Asch et al., 2009). The annealing temperature (Ta 57 st. C) and amplification conditions were established experimentally. The sequences were obtained using Sanger sequencing method performed by an external laboratory service.