Optimization of DNA from Musk Deer (Moschus chrysogaster) Hair Follicle: extraction, PCR Amplification for discriminatory analysis of RAPD markers

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Optimization of DNA from Musk Deer (Moschus chrysogaster) Hair Follicle: extraction, PCR Amplification for discriminatory analysis of RAPD markers

Inayat-Ullah Malik, Fakhar-i-Abbas, Muhammad Sajid Nadeem, Ghazala Kaukab, Zahid Sharif Mirza
Int. J. Biosci.13( 2), 257-264, August 2018.
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Of 62 hair follicle samples of Himalayan musk deer ((Moschus chrysogaster) collected by hair-snares appreciable quantity of DNA was isolated from 60.70%. Largest quantity of DNA (220.11±15.09ng/µL) was obtained by modified phenol-chloroform method, followed by phenol-chloroform (199.13±11.09ng/µL) and Qiagen kit (146.34 ±20.42ng/µL) methods. The qualities of extracted DNA by Qiagen kit and modified phenol-chloroform method were not significantly different within (paired t =1.68, df =33, P≥ 0.05; paired t=1.75, df=33, P≥0.05) but significantly higher (paired t=2.56, df=33, P≤0.05) than phenol-chloroform. Of 35 RAPD markers tested, 29 amplified. Optimal conditions varied for responding for PCR-amplification, as optimum concentration of: MgCl2; ranged minimum 1.5mm, 4 (13.79%) to maximum 2.5mm (6.89%), template DNA; ranged 30-50ng/µL, primer; majority,0.25 (16, 55.17%) to 0.15 (6, 20.68%), by using of A PCR profiles (Ta=26oC -37oC) for better and consistent amplification. Polymorphic information content (PIC) found higher (FA-18; 0.54) and lower (FA-9; 0.07) with resolution power (Rp) ranged 13.14-32.22. RAPD marker loci and molecular indices suggested significant correlation between PIC and Rp; (r2=0.95; P≤0.01), followed MI and PIC; (r2=0.67; P≤0.05) and MI and Rp (r2=0.63: P≤0.05). The probability of identical by chance ranged between 0.0012 (OPA-20) and 0.0357 (FA-14), and total probability by multiplying of 4.234 × 10-61. However, discriminatory indices of RAPD markers are indicative of high resolution and have potentials of significance, for using in DNA finger printing which can be employed for efficient identification, conservation and breeding approaches of musk deer.


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