Evaluation of different methods of DNA extraction from semen of buffalo (Bubalus bubalis) bulls

Abstract

Microbes excreted in the semen of infected or carrier bulls can be disseminated to susceptible animals through artificial insemination. The polymerase chain reaction (PCR) has been employed successfully to detect infectious agents in tissues and body fluids. PCR inhibitors present in the semen pose serious problems in detection of microorganisms by inhibiting the amplification of the target DNA template. These inhibitors need to be removed completely during DNA extraction to amplify the target sequences in semen by PCR. DNA was extracted using seven different protocols from semen of buffalo bulls, and the quantity and quality was evaluated spectrophotometrically. Chelex-100 and Qiagen modified methods for extraction of DNA from semen were found to be superior qualitatively as compared to the other methods. In the Qiagen modified protocol, the semen was treated with two extra buffers containing EDTA to chelate the metals. Additional treatment of semen with proteinase K was included to completely degrade cellular proteins. DNA extracted by the phenol-chloroform and CTAB methods yielded high value of residual RNA and other contaminants. The chelex-100 method has the potential advantage of requiring a smaller volume of semen to extract good quality of DNA.