ABSTRACT
BACKGROUND: Nowadays, definitive diagnosis of numerous diseases is based on the genetic and molecular findings. Therefore, preparation of fundamental materials for these evaluations is necessary. Deoxyribonucleic acid (DNA) is the first material for the molecular pathology and genetic analysis, and better results need more pure DNA. Furthermore, higher concentration of achieved DNA causes better results and higher amplifying ability for subsequent steps. We aim to evaluate five DNA extraction methods to compare DNA intimacy including purity, concentration, and amplifying ability with each other. MATERIALS AND METHODS: The lymphoid tissue DNA was extracted from formalin-fixed, paraffin embedded (FFPE) tissue through five different methods including phenol-chloroform as the reference method, DNA isolation kit (QIAamp DNA FFPE Tissue Kit, Qiagen, Germany), proteinase K and xylol extraction and heat alkaline plus mineral oil extraction as authorship innovative method. Finally, polymerase chain reaction (PCR) and real-time PCR method were assessed to compare each following method consider to DNA purity and its concentration. RESULTS: Among five different applied methods, the highest mean of DNA purity was related to heat alkaline method. Moreover, the highest mean of DNA concentration was related to heat alkaline plus mineral oil. Furthermore, the best result in quantitative PCR was in proteinase K method that had the lowest cycle threshold averages among the other extraction methods. CONCLUSION: We concluded that our innovative method for DNA extraction (heat alkaline plus mineral oil) achieved high DNA purity and concentration.
ABSTRACT
BACKGROUND: The aerobic Actinomycetes are a large group of soil-indwelling bacteria that are distributed in world-wide. These Gram-positive bacteria are most commonly associated with opportunistic infections in both immunocompromised and immunocompetent hosts. MATERIALS AND METHODS: In this study, three phenotypic and deoxyribonucleic acid (DNA) extraction methods for isolation and identification of Nocardia genus were compared. Samples were taken in five different locations of Isfahan's suburb from hospitals area, parks, agricultural lands, gardens, arid lands with different soil temperature and pH. RESULTS: In this study, showed that slip-buried-method was better than two other phenotypic methods; 14 out of 70 soil samples (20%) were positive for Nocardia spp. DNA of positive samples were extracted with three techniques and DNA extraction by microwave technique was better than others. This technique was confirmed with observation of DNA bands on 1% agarose gel. CONCLUSIONS: These bacteria are important in immune deficient patients such as cancer patients, transplant recipients, tuberculosis; acquired immunodeficiency syndrome etc., Their affluence is unsteady in different zones of the world. In this study, among the three phenotypic methods for the isolation of Nocardia slip-buried method was better than other methods. Among DNA extraction techniques, DNA extraction by microwave method would be selective method for DNA extraction of Nocardia spp. compared with others techniques.
ABSTRACT
SuperparamagneticSiO2@Fe3O4microspherewithcore-shellstructurewaspreparedbythe method of hydro-thermal and St?ber method, and modified by (3-aminopropyl) triethoxysilane ( APTES) . The obtained amino-Fe3 O4@SiO2 microsphere with controlled surface charge properties was characterized by SEM and TEM, and the surface electric property of amino-microsphere was investigated by Zeta potential measurement. The microsphere was used to extract DNA from human blood, and the solid phase extraction method by microspheres was developed. The mechanism of action between Aminated-Fe3 O4@SiO2 microsphere and DNA was explored, and the gel electrophoresis and PCR test were done on the extraction product. The result showed that genomic DNA with high purity was successfully extracted from whole blood by using amino-Fe3 O4@SiO2 microsphere, with the extraction rate about 70%, the saturated adsorption capacity for DNA was about 40 ng/μg, and the elution could be directly used for further bio-analysis.