The last aqueous phase was mixed with two-thirds volume of isopropanol and stored at −20 °C for at least 2 h to precipitate the DNA, then centrifuged at 4000 rpm for 15 minutes. The nucleic acid precipitate was washed with 70% ethanol, air dried, and suspended in 50 μl of TE buffer. DNA was treated with RNaseA (Quiagen, USA) for eradication of RNA followed by two washings with chloroform:iso-amyl-alcohol (24:1; v/v) before actual use. Subsequently, quality and quantity were checked by running the dissolved DNA in 0.8% agarose gel and uncut λ DNA (Bangalore Genei, Bangalore, India) of known concentration. The extracted DNA was diluted in ddH2O to 50 ng/μl and subjected
to RAPD-PCR analysis. Eighty five 10-base primers (Operon Technologies, Alameda, USA) were used for polymerase chain reaction (PCR) for screening of known sex to ascertain their potential of clear amplification in polymorphism and also the Transferase inhibitor reproducibility. The RAPD-PCR reactions were performed in 25 μl volumes in 100 μl PCR tubes (Tarson Pvt., Ltd., India). The reaction mixture contained 30 ng of template DNA, 1× amplification buffer (10 mM of Tris–HCl – pH 8, 50 mM of KCl, 1.8 mM of MgCl2 and 0.01 mg/ml gelatine), 2.5 mM each of dCTP, dGTP, dATP, and dTTP, 5 pM primers and 1 U Taq DNA
polymerase (Bangalore Genei, Pvt., Ltd., India). The reactions were performed in a Master Cycler Gradient 5331 (Eppendorf version 2.30. 31-09, Germany) with an initial denaturation step at 94 °C for 4 minutes, followed by 35 cycles of 94 °C for 1 minute, 37 °C for 1 minute, 72 °C for 2 minutes. The final extension Forskolin concentration step was at 72 °C for 10 minutes. The reactions were then cooled and held at 4 °C. The RAPD-PCR products were separated on 1.5% (w/v) agarose (Sigma–Aldrich, USA) gel at 5 V/cm in 1 × TBE (89 mM Tris–HCl, 89 mM boric acid and 2 mM EDTA, pH 8.0) buffer. The agarose gels were stained with 0.5 μg ml−1 ethidium Thiamet G bromide visualized under UV light and photographed on a digital gel-documentation system (SYNGENE). The molecular weights of the RAPD amplicons were estimated with a 100 bp DNA ladder (New England). A set of 85 decamer primers
were used to amplify the genomic DNA of male, female, and hermaphrodite individuals of which 16 primers showed reproducible results. Five primers OPU-10 (5ACCTCGGCAC3), OPD-19(5CTGGGGACTT3), OPU-19(5GTCAGTGCGG3), OPS-05(5TTTGGGGCCT3) and OPW-03(5GTCCGGAGTG3) produced unique amplicon for sex differentiation. Among these five decamer primers three primers, OPU-10, OPD-19, and OPU-19 showed sex specificity of male, female and hermaphrodite respectively. The primer OPU-10 produced a unique band in male individual DNA which was absent in female and hermaphrodite in the region above 1 kb DNA marker banding pattern (Fig. 1a). OPD-19 primer produced 350 bp unique amplicon in female individual’s DNA that was completely absent in male and hermaphrodite (Fig 1b).