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Abstract <p>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, <i>Taq</i> 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 MgCl<sub>2</sub>, 2 µL of 2.5 mM dNTPs, 4 µL of primer (10 picomoles/µL), 0.25 µL of 5U <i>Taq</i> 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.</p>

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Optimization of RAPD-PCR Assay for Assessing Genetic Variability in Brachyuran Crab Populations from Freshwater Ecosystems

  • 期刊论文

  • 发表日期：

    2025年9月4日

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  • Menakshi Dhar,
  • Seema Langer

摘要

Abstract

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.