Fe₃O₄@Al³⁺ For DNA Extraction And Its Application

With the demands of molecular biology biotechnology such as gene identification and family analysis,rapid nucleic acid extraction is increasingly important,especially during epidemic outbreaks.In this study,an Al³⁺-modified magnetic Fe₃O₄submicron particle(Fe₃O₄@Al³⁺)was prepared as a nucleic acid separation medium for rapid DNA extraction through metal chelation.The specific work is as follows:1.Submicron Fe₃O₄was prepared by solvothermal method.After epoxidizing,linking iminodiacetic acid,and chelating Al³⁺in turn,monodisperse superparamagnetic spherical submicron Fe₃O₄@Al³⁺about200 nm was obtained with high magnetic saturation intensity.（73.52emu/g）,good magnetic responsiveness,and the preparation process does not involve polymerization,so it will not agglomerate.Fe₃O₄@Al³⁺was used for DNA extraction studies.After optimization,it was determined that the optimal adsorption conditions for Fe₃O₄@Al³⁺extraction of E.coli plasmid DNA were 25°C,p H=4 adsorption for 5 min,and the maximum adsorption capacity was 7.41 mg/g;the elution conditions were3%eluate concentration,After 6 min of elution,the elution rate was91.23%,the extraction capacity was 6.32 mg/g,and the OD260/280 and OD260/230 of the obtained DNA solution were 1.93 and 2.02,respectively.The extracted DNA was used for PCR amplification with good fidelity.The ability of Fe₃O₄@Al³⁺to extract E.coli plasmid DNA was positively correlated with the amount of chelated Al³⁺.The mechanism of Fe₃O₄@Al³⁺for the extraction of E.coli plasmid DNA is the metal chelation between Al³⁺and the O atom on the phosphodiester bond of nucleic acid,etc.,the extraction capacity is high,the adsorption process does not need to add binding solution,and the washing does not need to use organic reagents such as ethanol,it only takes 15 minutes to complete the extraction of E.coli plasmid DNA,which greatly shortens the extraction time.2.Fe₃O₄@Al³⁺was used for the extraction of phage genomic DNA,and used for the detection of phage Ec NP1 content by q PCR dye method.The established standard curve:y=-3.1175x+37.545,R²=0.9987,with a good linear relationship,the detection linear range is 1.47×10¹～1.47×10⁷copies/μL,the detection limit is 7.3 copies/μL,and can be used to detect phage content in wastewater.3.Fe₃O₄@Al³⁺was used to extract soil bacterial metagenomic DNA and applied to soil bacterial 16Sr DNA diversity analysis.763,1007,and811 genera were detected in 3 different soil samples,which were mainly distributed in 4 phyla and 14 genera,of which the highest relative abundance was Proteobacteria.There are absolutely dominant species,two of which are Sphingomonas,which are obligate aerobic gram-negative bacteria with extensive metabolism of aromatic compounds,and the other is Gp4,which is Acidobacter An oligotrophic bacteria of the phylum.The novel monodisperse nucleic acid extraction medium Fe₃O₄@Al³⁺prepared in this study has a good application prospect in rapid nucleic acid extraction,phage detection and soil microbial diversity analysis.