OPTIMIZATION TECHNIQUE FOR GENOMIC DNA ISOLATION FROM BOVINE SPERM

Abstract

The bull sperm cells exhibit specific structures that protect their DNA, and therefore, the standard techniques used for somatic DNA isolation are not efficient. Thus, our objective was to design an inexpensive, quick and efficient bull sperm DNA isolation, under the hypothesis that the appropriate combination of chemical agents and a set of alternatives would allow us to design an improved method to extract DNA from bull sperm to be used in PCR assays. A series of replicates obtained from five ejaculates were used to evaluate six methods to extract bull sperm DNA: 1) the conventional method using proteinase K and phenol-chloroform; 2) papain, DTT, DMSO, CTAB, and SDS method (to solubilize the perinuclear theca and to replace phenol-chloroform); 3) the nonionic detergent Brij 36-T method, to remove the plasma membrane; 4) DTT and heparin method, for nuclear chromatin de-condensation; 5) SDS method and; 6) CTAB method, to remove the plasma membrane. DNA samples were analyzed spectrophotometrically to determine the DNA concentration and purity, then used at 200, 100, 50, 25, and 12.5 ng for PCR assays. To evaluate repeatability of the method, replicates of the most efficient procedure were performed. Results showed that the best method for bull sperm DNA extraction was the use of CTAB as a membrane removal agent; papain, DTT and DMSO as lysis buffer and DTT, heparin, and SDS as decondensing solution, achieving concentration ranging from 63 to 154 ng µL^-1 and optimal quality (A₂₆₀/A₂₈₀= 1.75 and 1.73) in fresh and frozen semen extracting, respectively. In conclusion, considering the structural characteristics of the sperm cell, we designed a method simple and efficient for extracting bull sperm DNA without limitations in PCR. Also, the whole procedure can be done in a short period of time without the need to employ phenol-chloroform and proteinase K treatment.