Optimization of RAPD-PCR Assay for Assessing Genetic Variability in Brachyuran Crab Populations from Freshwater Ecosystems
摘要
Genetic analysis of any species depends upon the yield of DNA samples. In the present study, randomly amplified polymorphic DNA (RAPD-PCR) was optimized for the crab species inhabiting the freshwater ecosystems. In the initial step, the optimal concentration of PCR components and cycling conditions were standardized by altering the parameters of PCR reaction such as PCR buffer, magnesium chloride, dNTPs, primer, Taq polymerase, template DNA, denaturation time, annealing temperature and time, and volume of the reaction. Results indicated that the RAPD-PCR optimization system was 2 µL of 10X PCR buffer with 1.5 mM MgCl2, 2 µL of 2.5 mM dNTPs, 4 µL of primer (10 picomoles/µL), 0.25 µL of 5U Taq polymerase, 1 µL of template DNA (50 ng/µL) and nuclease-free water to make the total volume of 20 µL with denaturation time of 1 min at 94°C and an annealing temperature of 35°C. The optimized PCR and its programming conditions were then used to study the genetic diversity within the populations of freshwater crab, found in the different locations of the Jammu region. Assessment of genetic diversity among any species or the population of the species is essential for aquaculture practices.