METHOD FOR ASSESSING INTENSITY OF PHOTOSYNTHESIS OF GRAPES USING RELATIVE COPY NUMBER OF CHLOROPLAST DNA Russian patent published in 2024 - IPC F26B9/06 

FIELD: biotechnology.

SUBSTANCE: invention is intended for estimation of intensity of photosynthetic processes using estimation of relative copy number of RPS16 gene of chloroplast DNA of grapes by method of Real Time-PCR. Method includes light exposure on sample of analyzed medium, wherein fresh leaves of grapes were used for extraction of total DNA, total genomic DNA from 5–10 mg of plant tissue of each sample was extracted using modified DNA extraction procedure, quantitative PCR (qPCR) was carried out using an automatic analyzer Light-Cycler 96 with initial denaturation at 95°C for 5 minutes—1 cycle, then 45 denaturation cycles at 95°C for 10 s, annealing at 58°C for 25 seconds and elongation at 72°C for 25 seconds, determining the levels of DNA of the organelles in relation to the target nuclear gene, the amount of the obtained DNA was measured by a fluorimetric method and using a reagent kit, quantitative RT-PCR reaction was carried out in 20 mcl of a reaction mixture consisting of 10 mcl of LightCycler 480 SYBR Green I Master Mix, 10 ng of total DNA 5 mcl, 3 mcl of water and 1 mcl of the corresponding primers, determining the relative copy number of the chloroplast DNA rps16 gene using the chromosomal DNA GAPDHb gene as a reference, for quantitative assessment of relative copy number of mitochondrial DNA, algorithms 2^−DCt and 2^−DDC_t were used, analysis of primary data was carried out using a software product of an amplifier, relative copy number of mitochondrial gene rps16 was calculated by formula 2^−ΔCt separately for experimental and control samples, K=K_exp/K_normal.

EFFECT: obtaining more accurate and specific assessment of intensity of photosynthetic processes.

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