Optimization and modification of sperm cells genomic DNA extraction protocol using Qiagen DNeasy® Blood & Tissue Kit for qPCR based sex validation techniques

Abstract

The effectiveness of PCR-based molecular techniques is closely tied to the quality of DNA template extraction. Obtaining high-quality DNA is essential for ensuring successful amplification and producing accurate, reliable results. This is because the purity and integrity of the DNA significantly impact the sensitivity and specificity of the PCR process. Conversely, suboptimal DNA extraction can introduce contaminants or degrade the DNA, which may interfere with amplification, leading to incomplete, erroneous, or failed results. Therefore, optimizing the extraction process is critical for the overall success of PCR-based assays. The current study evaluated three different DNA extraction methods to determine their effectiveness in extracting DNA from Vrindavani bull semen samples. The goal was to identify the most efficient method for obtaining high-quality DNA suitable for molecular analyses, such as PCR. Three methods include the real gene tissue kit, the Qiagen kit, and the modified Qiagen kit. The assessment focused on the quality, purity, and yield of the DNA extracted using each procedure. The evaluation of DNA quality and quantity from the various extraction methods showed notable differences among the three approaches. The modified Qiagen DNeasy Blood and Tissue Kit proved to be the most suitable method for extracting high-quality DNA in sufficient amounts from Vrindavani bull semen samples.