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Sazonova et al. Threshold heteroplasmy levels of atherogenic mutations

mass index > 30 kg/m ); untreated high blood pressure; the DNA samples were stored at -20 °C. For operating
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cigarette smoking; pulmonary embolism or deep vein with a collection of DNA samples, the samples were
thrombosis [20,21] . diluted in TE buffer to a concentration of 0.03 μg/μL.

The presence of increased IMT CA or atherosclerotic The concentration of DNA solution in ng/μL was
plaques was detected by high-resolution B-mode measured using IMPLEN NanoPhotometer
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ultrasound using a SSI-1000 scanner (SonoScape, nanospectrophotometer with the use of a
China) equipped with a 7.5-MHz linear array probe. LabelGuard™ microtubule in “DS DNA” mode at a
Carotid ultrasonography was performed by the same wavelength of 260 nm [16,17] .
researcher throughout the study. The far walls of the
right and left common carotid arteries, the bifurcation, The DNA samples of the study participants were used
the internal and the external carotid arteries were for carrying out the polymerase chain reaction (PCR)
visualized. The c-IMT of the first centimeter of the of fragments containing the region of 11 investigated
common carotid artery was measured in three different mutations [11-15] .
projections (anterior, posterior and lateral), and
analyses carried out with the use of dedicated software Electrophoresis of isolated DNA samples and PCR-
M’Ath (Metris, SRL France). The average value of these fragments was carried out in a horizontal apparatus of
measures was considered as an integral indicator of Helicon company in agarose gel using 0.5 × tris-borate-
intima-medial thickness [22-25] . The measurements were EDTA (TBE) buffer. The concentration of agarose
carried out in accordance with the Mannheim criteria (“Fluka”) was 0.8% (for DNA samples) and 1.5-2.0% (for
and the criteria of the investigation Improve [26,27] . PCR-fragments). The gel was stained by the addition
of an ethidium bromide solution (0.5 μg/mL). As a
Materials colorant, a solution of bromophenol blue (1 μL for 10 μL
The materials for the study were blood leukocyte of sample) was used [16-18] . The composition of 10 × TBE:
samples of participants. Blood for genetic analysis TrisHCl (108 g), boric acid (55 g), 0.5 mol/L EDTA, pH
was taken after an overnight fast in the amount of 9 mL 8.0 (40 mL per 1 liter of buffer).
from the ulnar vein in a 10 mL plastic tube containing
sodium salt of Na -ethylenediaminetetraacetic (EDTA) For the evaluation of molecular weight of the
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acid as an anticoagulant. A mother liquor of 0.1 mol/L investigated PCR fragments, DNA markers of 1 Kb
Na-EDTA in water (pH 8.0) was used, to which fresh (13 fragments from 0.25 to 10 Kb) and 100 bp (10
blood was added in a ratio of 9:1 to obtain a final Na- fragments from 100 to 1,000 bp) were used [16-18] .
EDTA concentration of 10 mmol/L. Samples were
stored at -20 °C. For conducting PCR, DNA with a concentration of
0.1 μg/mL diluted with μQ and primers at a concentration
Procedure of 10 pmol/μL were taken [16-18] . The reaction mixture and
It is noteworthy that in previous research from our PCR conditions were the following: µQ (H O): 4.6 μL;
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laboratory, we haven’t found any significant differences a mixture of dNTPs 10×: 2 mmol/L deoxyadenosine
in heteroplasmy levels of the investigated mutations triphosphate, 2 mmol/L dTTP, 2 mmol/L dGTP, 2 mmol/L
between leukocytes and platelets (blood cells having dCTP: 4 μL; 10× PCR buffer (16.6 μmol/L (NH ) SO , 67
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mitochondrial genome), so DNA samples for this study mmol/L Tris-HCl (pH 8.8): 4 μL; MgCl : 25 mmol/L: 4 μL
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were isolated from whole blood [16-19,28] . This method (at the required concentration of 2.5 mmol/L); 2.4 μL
was developed in our laboratory on the basis of (at the required concentration of 1.5 mmol/L); Taq
technology of Maniatis [29] . For DNA isolation we used polymerase: 1.33 μL; Matrix DNA: 4 μL; Primer F (+):
a lysis buffer (0.32 mol/L sucrose; 5 mmol/L MgCl ; 2.7 μL; Primer R (-): 2.7 μL. The reaction was carried
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100% Triton X-100; 0.01 mol/L Tris-HCl, pH 7.6), and out in 40 μL of the reaction mixture [16,17] .
then deproteinization buffer (25 mmol/L EDTA pH 8.0;
75 mmol/L NaCl) and proteinase K solution (20 mg/mL One of the PCR primers was biotinylated with the
DNA purification from admixtures was carried out aim of the subsequent pyrosequencing of the PCR
using phenol and chloroform. Precipitation of DNA was fragment. The study was carried out using amplifier
carried out using isopropanol, followed by washing in “PTC DNA Engine 200”. After that pyrosequencing of
ethanol. The DNA precipitate was dissolved in 300 μL PCR fragments was conducted using the apparatus
of TE buffer (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L (PSQ96MA) [30-34] . The pyrosequencing reaction included
EDTA). The DNA concentration in the obtained sample 4 stages [30-34] .
was identified by nanospectrophotometer IMPLEN
NanoPhotometer . After measuring the concentration, Stage 1: the sequence primer was hybridized to a
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184 Vessel Plus ¦ Volume 1 ¦ December 28, 2017