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Sazonova et al. Vessel Plus 2019;3:8 I http://dx.doi.org/10.20517/2574-1209.2019.01 Page 3 of 7

Table 1. The size of DNA amplicons and primers for PCR
Mutation Primers Size of DNA amplicons
m.12315G>A F: bio-CTCATGCCCCCATGTCTAA(12230-12249) 108 bp
R: TTACTTTTATTTGGAGTTGCAC(12337-12317)
m.652delG F: TAGACGGGCTCACATCAC(621-638) 467 bp
R: bio-GGGGTATCTAATCCCAGTTTGGGT(1087-1064)
m.3336T>C F: bio-AGGACAAGAGAAATAAGGCC(3129-3149) 294 bp
R: ACGTTGGGGCCTTTGCGTAG(3422-3403)
m.14459G>A F: CAGCTTCCTACACTATTAAAGT(14303-14334) 209 bp
R: bio-GTTTTTTTAATTTATTTAGGGGG(14511-14489)
m.5178C>A F: bio-GCAGTTGAGGTGGATTAAAC(4963-4982) 383 bp
R: GGAGTAGATTAGGCGTAGGTAG(5366-5345)
m.13513G>A F: CCTCACAGGTTTCTACTCCAAA(13491-13512) 335 bp
R: bio-AAGTCCTAGGAAAGTGACAGCGAGG(13825-13806)
m.652insG F: TAGACGGGCTCACATCAC(621-638) 467 bp
R: bio-GGGGTATCTAATCCCAGTTTGGGT(1087-1064)
m.3256C>T F: bio-AGGACAAGAGAAATAAGGCC(3129-3149) 294 bp
R: ACGTTGGGGCCTTTGCGTAG(3422-3403)
m.15059G>A F: bio-CATTATTCTCGCACGGACT(14671-14689) 450 bp
R: GCTATAGTTGCAAGCAGGAG(15120-15100)
m.1555A>G F: TAGGTCAAGGTGTAGCCCATGAGGTGGCAA(1326-1355) 379 bp
R: bio-GTAAGGTGGAGTGGGTTTGGG(1704-1684)
m.14846G>A F: bio-CATTATTCTCGCACGGACT(14671-14689) 450 bp
R: GCTATAGTTGCAAGCAGGAG(15120-15100)

bp: base pairs

compare the samples of patients with cardiac angina and conventionally healthy study participants more
correctly, the samples were composed so that they did not have significant differences in age and sex.

The work was conducted in complance with the Declaration of Helsinki. The study protocol has been
accepted by Ethics Community of National Medical Research Center of Cardiology, and all subjects signed
an informed consent for inclusion in the research.

DNA from blood leukocyte samples was isolated using a phenol-chloroform method [13,14,23-25] . DNA
amplicons containing the investigated regions of 11 mitochondrial genome mutations (m.12315G>A,
m.652delG, m.5178C>A, m.14459G>A, m.3336T>C, 652insG, m.3256C>T, m.1555A>G, m.15059G>A,
m.13513G>A, m.14846G>A) were pyrosequenced. The heteroplasmy level of mtDNA mutations was analyzed
using a method developed by our laboratory.

The size of DNA amplicons and primers for PCR are listed in Table 1 [13-16,20] .

In order to be able to perform pyrosequencing of DNA amplicons, one of the primers for PCR was
biotinylated.

The total volume of PCR reaction mixtures for each sample was 30 mL. The composition of the reaction
mixture for PCR [13-16,20] : 0.4-0.6 mg mitochondrial DNA, 0.3 pmol/L of each primer, 200 mmol/L of each
deoxyribonucleotriphosphate, 16.6 mmol/L (NH ) SO , MgCl (1.5 mmol/L for mutations m.14846G>A,
4 2
4
2
m.15059G>A and m.14459G>A; 2.5 mmol/L for the rest of investigated mutations), 67 mmol/L tris-HCl (pH
8.8), and 3 units of Taq-polymerase.
In PCR, the following annealing temperature was used for the primers [13-16,20] :
1. For mutations m.3336T>C, m.14846G>A, m.13513G>A, m.15059G>A and m.3256C>T - 55 °C;
2. For mutations m.5178C>A, m.652delG and m.652insG - 60 °C;
3. For mutations m.12315G>A, m.14459G>A and m.1555A>G - 50 °C.