Bìol. Tvarin, 2017, Volume 19, Issue 2, pp. 56–63

OPTIMIZATION OF POLYMERASE CHAIN REACTION CONDITIONS FOR STUDIES OF PADDLEFISH (POLYODON SPATHULA) MICROSATELLITE DNA

Kh. Kurtа, O. Malysheva, V. Spyrydonov

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Ukrainian Laboratory of Quality and Safety of Agricultural Products,
National University of Life and Environmental Sciences of Ukraine,
7 Mashynobudivnykiv str., Chabany village, Kyiv region, 08162, Ukraine, This email address is being protected from spambots. You need JavaScript enabled to view it.

Paddlefish (Polyodon spathula) is a valuable object of artificial breeding and raising in fish farms in Ukraine. The study of genetic variability of paddlefish populations using new molecular-genetic approaches with microsatellite DNA markers is an important task for optimization of reproduction and preservation of paddlefish gene pool, effective cultivation and production of valuable fish products.

The important conditions for successful polymerase chain reaction (PCR) are optimal temperature and duration of each cycle, components of the reaction mixture and their concentration. It should be noted that efficiency of PCR-reaction mainly depends on DNA-matrix concentration, concentration of magnesium ions (Mg2+) and primer annealing temperature.

The objective of the studies was the optimization of PCR conditions and their assessment by selection of microsatellite DNA markers, amplification conditions and composition of the reaction mixture for maximum specificity and efficiency of paddlefish (Polyodon spathula) microsatellite DNA amplification.

Based on comparative analysis of previous studies of microsatellite DNA markers polymorphism of different paddlefish populations we have determined that Psp12, Psp21, Psp26, Psp28 DNA-markers are the most informative for further research. The optimal PCR-amplification conditions, namely a temperature regime and cycles duration of polymerase chain reaction were selected for mentioned DNA-markers. The optimal composition and concentration of components of the PCR stock mixture were defined. According to the results of the studies we have defined the effective annealing temperature of primers (TA) is 56 °C, the optimal concentration of each primer is 5 pM/mm3 and the final concentration of magnesium ions (Mg2 +) is 1.5 mM. The total volume of the final PCR mixture is 15 mm3. The results of PCR conditions optimization for paddlefish microsatellite DNA loci allow us to conduct the further studies of the genetic structure for the evaluation of genetic polymorphism of this species in a modern aquaculture.

Keywords: PADDLEFISH, MICROSATELLITE, DNA-MARKERS, POLYMERASE CHAIN REACTION, GENOTYPING, POLYMORPHISM, GENETIC STRUCTURE

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