Pengembangan metodologi ekstraksi dan purifikasi DNA dari bulu burung Perkutut (Geopelia striata) untuk pemanfaatan studi sexing dan keragaman genetik

Abstract

In the molecular studies of birds, the total DNA obtained from the extraction and purification of blood. Bird turtledove is very small and easy to stress so it is very risky if the blood is taken. Plumage may be other alternatives in obtaining DNA because it contains epithelial cells at their bases. The purpose of this research was to obtain a methodology of extraction and purification of DNA from the feathers of birds turtledove (Geopelia stiata) to study the utilization of sexing and genetic diversity. The method used consists of three kinds: 1) Digestion buffer-based method, 2) modification method using cetyl Trimethyl Ammonium bromide (CTAB buffer), and 3) the method of using tablets InhibitEX in CTAB buffer modification. The third method were divided into two treatments, the tablet was added before the addition of a solution of phenol or InhibitEX Before Phenol (IBP) and, the tablet was added after the addition of a solution of phenol or treatment InhibitEX After Phenol (IAP). The treatment period of soaking the samples in low-TE solution was during in 3 days and 14 days. The quality of DNA bands seen from the results of electrophoresis and spectrophotometer then compared with the control DNA from blood. Based on the purification result, the DNA was not obtained from Digestion buffer method. But, the DNA was obtained in CTAB buffer method, and the method using tablets InhibitEX been obtained DNA. Value averaging the results of the second method of DNA purity was lower (1.156) compared with the third method that is 3IBP (1.321) and 3IAP (1.551). PCR process was conducted as part to evaluate whether the DNA template (DNA template) from the extraction of fur can be amplified without a hitch or not. Based on the PCR test, 3IAP method has a high percentage (100%) compared with 3IBP method (50%) and method 2 (10%).

Dalam studi molekuler burung, DNA total didapatkan dari hasil ekstraksi dan purifikasi darah. Burung perkutut berukuran sangat kecil dan mudah stres sehingga sangat beresiko bila diambil darahnya. Bulu burung dapat menjadi alternatif lain dalam memperoleh DNA karena mengandung sel epitel pada bagian pangkalnya. Tujuan dari penelitian ini adalah mendapatkan metodologi ekstraksi dan purifikasi DNA dari bagian bulu burung perkutut (Geopelia stiata) untuk pemanfaatan studi sexing dan keragaman genetik. Metode yang digunakan terdiri dari tiga macam yaitu: 1) metode berbasis Digestion buffer, 2) metode menggunakan Cetyl Trimethyl Ammonium Bromide (CTAB buffer), dan 3) metode menggunakan tablet InhibitEX pada modifikasi CTAB buffer. Metode ketiga dibagi menjadi dua perlakuan yaitu tablet ditambahkan sebelum penambahan larutan phenol atau InhibitEX Before Phenol (IBP) dan tablet ditambahkan setelah perlakuan penambahan larutan phenol atau InhibitEX After Phenol (IAP). Perlakuan masa perendaman sampel pada larutan low-TE selama 3 hari dan 14 hari. Kualitas DNA dilihat dari pita hasil elektroforesis dan spektrofotometer lalu dibandingkan dengan DNA kontrol dari darah. Berdasarkan hasil ekstraksi dan purifikasi, DNA tidak diperoleh pada metode Digestion buffer. Tetapi, DNA diperoleh pada metode CTAB buffer, dan metode menggunakan tablet InhibitEX. Nilai rataan kemurnian DNA hasil metode kedua lebih rendah (1,156) dibandingkan dengan metode ketiga yaitu 3IBP (1,321) dan 3IAP (1,551). Berdasarkan uji PCR, metode 3IAP memiliki persentase yang tinggi (100%) dibandingkan dengan metode 3IBP (50%) dan metode 2 (10%).