Development and evaluation of an efficient low-cost DNA extraction Kit at genomic and molecular biology laboratory, Makerere University.
Abstract
Nucleic acid extraction is a common yet critical task in the molecular biology laboratory. Also known as nucleic acid isolation or nucleic acid purification, removal of genetic material from the sample matrix is the first step in many genetic and genomic studies. Preparing high quality samples improves the likelihood of your downstream process. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Therefore, there is need to advance in development of DNA extraction kit. To identify and evaluate the most effective combination of readily available reagents for developing a low cost DNA extraction kit, to determine the yield and purity of the DNA extracts and to compare the performance of the kit in the extraction of DNA between Eschericia coli and Mycobacterium tuberculosis bacteria. Methodology: Eschericia coli and Mycobacterium tuberculosis samples were subjected to cell lysis using a combination of Tris-HCL (pH 8.0), EDTA, SDS, and proteinase K. DNA purification was performed using sodium chloride and ethanol, followed by DNA washing with ethanol and elution in nuclease-free water. The extracted DNA was quantified using spectrophotometric analysis, (Nano drop) and its purity was assessed by measuring the absorbance ratios. The results revealed significant differences in DNA yield and purity between E. coli and M. tuberculosis samples. E. coli consistently exhibited higher DNA yield and greater DNA purity compared to M. tuberculosis. The differences in DNA characteristics can be attributed to variations in cell wall composition and other biological factors specific to each bacterial species. There was variation in the performance of the DNA extraction kit between E. coli and M. tuberculosis, with good results obtained for E. coli samples. These findings highlight the importance of considering the specific characteristics of the target organisms when designing and optimizing DNA extraction protocols. The variations in DNA yield and purity between the two bacterial species have implications for future research involving these organisms.