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Sazonova et al. Threshold heteroplasmy levels of atherogenic mutations

single-stranded PCR amplicon, which is used as Then, a solution of streptavidin-sepharose was prepared
a template. The obtained fragment was incubated for 1 sample: 40 μL 2 × BB, 3 μL of streptavidin-
with the following enzymes: DNA polymerase, ATP- sepharose particles, 7 μL of μQ H O. After that, the
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sulfurylase, luciferase and apyrase; and it was mode in the computer program for pyrosequencing was
also incubated with the substrate: adenosine-5- set. Into each test tube, 50 μL of streptavidin-sepharose
phosphosulfate and luciferin. solution were added, previously shaking it with vortex.
Samples were put in a shaker for 5 min. At this time,
Stage 2: the first nucleotide was added to the 39 μL of 1 × AB and 3 μL of corresponding probe primer
reaction mixture. DNA polymerase completes it to with a concentration of 2 optical units were added to
the DNA strand, in accordance with the principle each well of the pyrosequencing dish and the prepared
of complementarity. In this case, the reaction is pyrosequencing dish was placed in the runners of a
accompanied by the release of pyrophosphate (PPi) vacuum sample preparation station. The dishes were
in an amount equimolar to the sum of the included placed into the appropriate sample niches in the sample
nucleotides. preparation station, the following reagents were poured
into all the baths: washing buffer (WB), 0.2 mol/L NaOH,
Stage 3: ATP-sulphurylase converts PPi to ATP in rectified alcohol 70%, μQ H O. The nozzle with filters
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the presence of adenosine-5-phosphosulfate. In this was placed into the dish with μQ H O and vacuum was
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case, luciferin is converted into oxyluciferin, which switched on at the sample preparation station. In 20 s
generates visible light in proportion to the amount of the nozzle was removed from the dish and dried in an
ATP. A pyrosequenator detects this light and converts upright position for 10 s. Then the nozzle with filters was
it into the corresponding peak on the pyrogram. The placed in the 96-well pyrosequencing plate with samples
height of each peak is proportional to the intensity of for 30 s. Then the filters with samples were lowered into
the flash of light, and, consequently, to the number of reagent dishes: rectified alcohol 70% (for 5 s), then into
analyzed nucleotides in the DNA strand. For example, 0.2 mol/L NaOH (for 5 s), then into WB (for 10 s) and
if there is one nucleotide C in a particular position of then into μQ H O (for 5 s). Then the nozzle with filters
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the DNA strand, a peak corresponding to a single flash was raised and held in the upright position for 10 s. After
will be seen on the pyrogram, and if there are three this procedure, a nozzle with filters was placed above
nucleotides C in succession, a triple flash will be seen the pyrosequencing plate (without touching the primer
(i.e. the peak will be three times higher). solution with the nozzle filters), the vacuum was turned
off and only after that the nozzle filters were lowered into
Stage 4: apyrase, a nucleotide-destroying enzyme, the plate. In 2 min the nozzle with filters was removed
constantly removes the remaining nucleotides which from the pyrosequencing plate. The pyrosequencing
were not attached and ATP. Afterwards another plate was placed on a thermostat at 80 °C for 2 min.
nucleotide is added to the reaction mixture. Before placing the plate into the sequencer, it was given
time to cool down. During the cooling of the plate, the
To carry out the above-described investigation, it was reagents E (enzyme) and S (substrate) were diluted with
necessary to conduct sample preparation: a mixture μQ H O, E, S, and individual dNTPs were poured into
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of primers for the sequence should be added to the the cuvette, according to the instructions, at the amount
studied single-chain biotinylated PCR-fragments. indicated for setting the mode for sequencing. After
Streptavidin-sepharose particles were attached to that, the cuvette with the reagents and the samples was
these biotinylated PCR-fragments, which makes it put into the sequencer and the program was launched.
possible to operate with single-strand PCR-fragments
using streptavidin interacting with biotin. The sequences of primers for pyrosequence were
given in the article of Sazonova et al. [16] Visualization
The algorithm of sample preparation for pyrosequencing of the results was carried out with the use of a
was as follows: (1) 2 × Binding buffer [per 100 mL of computer program, which was completed with the
the solution: С Н NO (0.121 g), NaCl (11.7 g), EDTA pyrosequenator. The heteroplasmy level of every
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(0.0292 g), Tween 20 (100 μL). PH was adjusted to 7.6, mitochondrial genome mutation was assessed on the
1 mol/L HCl]; (2) 1 × Annealing buffer [per 100 mL of the basis of the pyrogram of a DNA sample from each study
solution: С Н NO (0.242 g), C H MgO × 4H O (0.043 g). participant, using a formula previously developed by
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PH was adjusted to 7.6, 4 mol/L CH COOH]; (3) washing Sazonova et al. [16,17] with colleagues: P = (h - N)/(M - N)
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buffer [per 100 mL of solution: С Н NO (0.121 g). PH was × 100, where “P” is the heteroplasmy percentage of the
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adjusted to 7.6, 4 mol/L CH COOH]; and (4) 0.2 mol/L studied mutations; “h” is the height of the peak of the
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NaOH (per 100 mL solution: NaCl 0.8 g). investigated nucleotide; “N” is the height of the peak
Vessel Plus ¦ Volume 1 ¦ December 28, 2017 185