Abstract：
High-quality DNA is an important guarantee to start downstream experiments in many biological and medical research areas. Magnetic particle-based DNA extraction methods from blood mainly depend on electrostatic adsorption in a low-pH environment. However, the strong acidic environment can influence the DNA stability. Herein, a polydopamine-functionalized magnetic particle (PDA@Fe3O4)-based protocol was developed for DNA extraction from whole blood samples. In the protocol, Mg2+ and Ca2+ were utilized to bridge the adsorption of DNA by PDA@Fe3O4 via the metal-mediated coordination. Isopropanol was found to efficiently promote DNA adsorption by triggering the change of the conformation of DNA from B-form to more compact A-form. In 50 % isopropanol solution, the DNA adsorption efficiency was nearly 100 % in the presence of 0.5 mM Ca2+ or 1.5 mM Mg2+. The role of metal ions and isopropanol in DNA adsorption was explored. The protocol averts the strong acidic environment and PCR inhibitors, such as high concentrations of salt or polyethylene glycol. It demonstrates superiority in DNA yield (59.13 ± 3.63 ng μL-1) over the commercial kit (27.33 ± 4.98 ng μL-1) and phenol-chloroform methods (37.90 ± 0.47 ng μL-1). In addition, to simplify the operastion, an automated nucleic acid extraction device was designed and fabricated to extract whole genomic DNA from blood. The feasibility of the device was verified by extracting DNA from cattle and pig blood samples. The extracted DNA was successfully applied to discriminate the beef authenticity by a duplex PCR system. The results demonstrate that the DNA extraction protocol and the automated device have great potential in blood samples.
摘要：
高质量的DNA是许多生物和医学研究领域开展下游实验的重要保证。从血液中提取基于磁性颗粒的DNA的方法主要依赖于低pH环境中的静电吸附。然而,强酸性环境会影响DNA的稳定性。在这里，开发了一种基于聚多巴胺功能化磁性颗粒（PDA@Fe3O4）的方案，用于从全血样品中提取DNA。在协议中,利用Mg2和Ca2通过金属介导的配位来桥接PDA@Fe3O4对DNA的吸附。发现异丙醇通过触发DNA构象从B形式到更紧密的A形式的变化来有效地促进DNA吸附。在50%异丙醇溶液中,在0.5mMCa2或1.5mMMg2存在下，DNA吸附效率接近100％。探讨了金属离子和异丙醇在DNA吸附中的作用。该方案避免了强酸性环境和PCR抑制剂，如高浓度的盐或聚乙二醇。与商业试剂盒（27.33±4.98ngμL-1）和苯酚-氯仿方法（37.90±0.47ngμL-1）相比，它证明了DNA产量（59.13±3.63ngμL-1）的优越性。此外,为了简化操作，设计并制造了一种自动核酸提取装置，用于从血液中提取全基因组DNA。通过从牛和猪的血液样本中提取DNA，验证了该装置的可行性。通过双重PCR系统成功地将提取的DNA用于鉴别牛肉的真实性。结果表明，DNA提取方案和自动装置在血液样本中具有巨大的潜力。
